CN116059441A - Anisotropic nanofiber composite natural polysaccharide hydrogel and preparation method and application thereof - Google Patents
Anisotropic nanofiber composite natural polysaccharide hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedical engineering, and particularly relates to an anisotropic nanofiber composite natural polysaccharide hydrogel and a preparation method and application thereof. The hydrogel comprises S1 and S2: s1: an anisotropic nanofiber layer, the anisotropic nanofiber being prepared from the following raw materials: modified polysaccharide and synthetic polymer; s2: a modified polysaccharide layer; or a modified polysaccharide layer comprising a functional substance; the hydrogel has photocuring capability and printability through modified polysaccharide; the hydrogel has good mechanical properties through the anisotropic nanofiber, and simultaneously, the osteogenic differentiation of cells is inhibited; can be used for preparing biomedical materials with tissue repair function.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering, and particularly relates to an anisotropic nanofiber composite natural polysaccharide hydrogel and a preparation method and application thereof.
Background
Calcified heart valve disease (CAVD) is a common serious disease that jeopardizes the physical and mental health of humans, and there is currently no method to prevent and treat CAVD, and when patients develop end-stage CAVD characterized by small She Gaihua and severe stenosis, the only option is valve replacement. Mechanical and biological valves are two choices commonly used clinically at present, and have good and bad performances, but the common defects of the mechanical and biological valves are that the mechanical and biological valves cannot randomly develop and grow, and the mechanical and biological valves are especially inapplicable to infant patients with the heart which is not yet developed. The Tissue Engineering Heart Valve (TEHV) is characterized in that autologous seed cells are planted on degradable biological materials, and the heart valve with normal structure and function is finally formed along with the continuous proliferation of the seed cells, the secretion of extracellular matrix (ECM) and the gradual degradation and absorption of the biological materials, so that the heart valve has very important clinical application prospect.
The key to constructing TEHV is the scaffold on which seed cells survive, the mechanical properties close to soft tissue and good biocompatibility achieved by natural polysaccharide hydrogels, which have been widely used in recent years to prepare scaffolds, but are also reinforced in terms of mechanical properties and degradation rates, etc., furthermore, hydrogel polymer networks are generally isotropic, while many biological systems have well-defined layered structures with macroscopic anisotropy, such as heart valves, skin and articular cartilage. In heart valves, anisotropy plays a critical role in achieving heart contraction and relaxation, etc., and anisotropic structures have a great influence on proliferation, migration and differentiation of cells.
The hydrogel material based on natural polysaccharide such as gelatin is favorable for seed cell inoculation and growth in the aspects of biocompatibility, degradability, cell material interface, three-dimensional porous structure, plasticity and the like, and is an ideal tissue engineering matrix material. It has been demonstrated that fibroblasts, chondrocytes and osteoblasts can survive in gelatin-based hydrogels and form extracellular matrix, but their mechanical strength is to be improved. Numerous studies have shown that natural polysaccharide is an ideal tissue engineering hydrogel biomaterial, but it does not exhibit nonlinear mechanical properties critical to valve biomechanics and may enhance the characteristics of CAVD such as MSC osteogenic differentiation and ECM calcification. Thus, natural polysaccharide hydrogels such as gelatin need to be further modified or compounded with other materials to reduce the risk of potential calcification or leaflet retraction. In addition, the hydrogel scaffold can provide a microenvironment which is closer to the natural cartilage extracellular matrix for cell proliferation and differentiation, and is an ideal material for soft tissue repair. The traditional hydrogel scaffold manufacturing technology cannot realize individuation and complex geometric shapes, soft tissue engineering relates to multiple factors such as scaffolds, cell induction and factor stimulation, proper biomechanical environment and the like, and has high requirements on mechanical strength, degradation performance, scaffold geometric shapes and the like. 3D bioprinting has achieved significant results in the field of regeneration of implantable tissues as an emerging technology for tissue engineering. The main challenges facing the present are how to keep the cell high activity and produce the high-toughness hydrogel biological scaffold so as to meet the clinical application requirements. When the hydrogel is applied to 3D biological printing, biocompatibility and cross-linking coagulation in the printing process are required to be considered, so that the selection range is limited. Finding out a suitable printable hydrogel as a cell carrier to form 3D bio-printing "ink" is a key link for solving the current 3D bio-printing.
Disclosure of Invention
The object of the first aspect of the present invention is to provide an anisotropic nanofiber composite polysaccharide hydrogel.
The object of the second aspect of the present invention is to provide a method for preparing the anisotropic nanofiber composite polysaccharide hydrogel of the first aspect.
The object of a third aspect of the present invention is to provide the use of the anisotropic nanofiber composite polysaccharide hydrogel of the first aspect in the preparation of biomedical materials.
The object of the fourth aspect of the present invention is to provide a biomedical material.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided an anisotropic nanofiber composite polysaccharide hydrogel comprising S1 and S2:
s1: an anisotropic nanofiber layer, the anisotropic nanofiber being prepared from the following raw materials: modified polysaccharide and synthetic polymer;
s2: a modified polysaccharide layer; or (b)
A modified polysaccharide layer comprising a functional material;
the modified polysaccharide comprises at least one of methacryloyl modified polysaccharide and methacryloxypropyl modified polysaccharide;
the synthetic polymer comprises at least one of polycaprolactone, polylactic acid, polyethylene glycol, polybutylene terephthalate-adipate and polyglycolide;
The functional substance comprises at least one of a cell growth factor and a stem cell.
Preferably, the S1 and S2 are alternately laminated.
Preferably, the number of layers of the S1 is n, and the number of layers of the S2 is n+1 or n-1; further preferably, the number of layers of S1 is n, and the number of layers of S2 is n+1.
Preferably, n is 2, 3, 4, 5, or 6; further preferably, n is 2, 3, 4, or 5.
Preferably, the modified polysaccharide comprises a methacryloyl modified polysaccharide.
Preferably, the preparation method of the methacryloyl modified polysaccharide comprises the following steps: mixing the polysaccharide with a monomer containing a methacryloyl group, and reacting to obtain the methacryloyl modified polysaccharide.
Preferably, the polysaccharide comprises at least one of gelatin, hyaluronic acid, sodium alginate, carrageenan, dextran; further preferably, the polysaccharide comprises at least one of gelatin, hyaluronic acid, sodium alginate; still more preferably, the polysaccharide comprises one or two of gelatin, hyaluronic acid, sodium alginate; further, the composition is gelatin, hyaluronic acid, sodium alginate, a mixture of gelatin and sodium alginate, or a mixture of gelatin and hyaluronic acid.
Preferably, the mass-to-volume ratio (g: mL) of the polysaccharide to the monomer containing the methacryloyl group is (0.5-15): 7.5; further (1-10): 7.5.
preferably, the methacryloyl group containing monomer comprises at least one of methacrylic anhydride, glycidyl methacrylate; further comprising methacrylic anhydride.
Preferably, the polysaccharide is a polysaccharide solution.
Preferably, the polysaccharide solution has a pH of 7.5 to 10; further 8 to 9.
Preferably, the concentration of the polysaccharide solution is 0.5-30% in terms of mass-to-volume ratio (w/v, g/mL); further preferably, the concentration of the polysaccharide solution is 1 to 10% by mass volume (w/v).
Preferably, when the polysaccharide is gelatin, the concentration of the polysaccharide solution is 5-30% in terms of mass-to-volume ratio (w/v, g/mL); further preferably, the polysaccharide solution has a concentration of 5 to 20% by mass volume (w/v).
Preferably, the gelatin has a number average molecular weight of 30000D to 100000D; further 30000D to 80000D.
Preferably, when the polysaccharide is hyaluronic acid or sodium alginate, the concentration of the polysaccharide solution is 0.5-2% in terms of mass-to-volume ratio (w/v, g/mL); further preferably, the concentration of the polysaccharide solution is 1-2% by mass volume (w/v).
Preferably, the hyaluronic acid has a number average molecular weight of 1,000 to 1,800,000; further 1 200 000 to 1,800,000D.
Preferably, the number average molecular weight of the sodium alginate is 30000D-100000D; further 60000D-100000D.
Preferably, the conditions of the reaction are: the pH=7.5 to 10, and the reaction is carried out for 4 to 8 hours at the temperature of 35 to 45 ℃; the method further comprises the following steps: the pH=8-9, and the reaction is carried out for 6-8 h at 35-40 ℃.
Preferably, the reaction further comprises the steps of dialysis and drying.
Preferably, the molecular weight cut-off in the dialysis is 3000-4000D; further 3000-3500D; and further 3500D.
Preferably, the functional substance comprises stem cells; further comprising mesenchymal stem cells.
Preferably, the mesenchymal stem cells have a density of 10 in the modified polysaccharide 5 ～10 8 cell/mL modified polysaccharide; further 10 6 ～10 7 cell/mL modified polysaccharide.
Preferably, the modified polysaccharide comprises at least one of methacryloyl group modified gelatin, methacryloyl group modified hyaluronic acid, and methacryloyl group modified sodium alginate; further preferably, the modified polysaccharide comprises a methacryloyl group modified gelatin, a methacryloyl group modified hyaluronic acid, a methacryloyl group modified sodium alginate, a mixture of a methacryloyl group modified gelatin and a methacryloyl group modified hyaluronic acid, or a mixture of a methacryloyl group modified gelatin and a methacryloyl group modified sodium alginate.
Preferably, the mixture of the methacryloyl group modified gelatin and the methacryloyl group modified hyaluronic acid has a mass ratio of 10 to 40:1, a step of; further 30-40: 1.
preferably, the mixture of the methacryloyl group modified gelatin and the methacryloyl group modified sodium alginate has a mass ratio of 10 to 40:1, a step of; further 10 to 20:1.
preferably, the degree of orientation of the anisotropic nanofibers is 0.112 to 0.125; further 0.113 to 0.118.
Preferably, the thickness of the anisotropic nanofiber is 0.1-1.3 mm; further 0.2-1.0 mm; and further 0.2 to 0.5mm.
Preferably, the thickness of the modified polysaccharide layer is 3-6 mm; further 4 to 5mm.
Preferably, the synthetic polymer comprises at least one of polycaprolactone, polylactic acid, polyethylene glycol, polybutylene terephthalate-adipate; further comprising at least one of polycaprolactone, polylactic acid, polyethylene glycol; further comprises polycaprolactone, polylactic acid or polyethylene glycol.
Preferably, the number average molecular weight of the polycaprolactone is 40000D-100000D; further 60000-65000D.
Preferably, the polylactic acid has a number average molecular weight of 10000D-100000D; further 40000D to 60000D.
Preferably, the polyethylene glycol has a number average molecular weight of 1000D to 20000D; further 6000D to 8000D.
Preferably, the mass ratio of the modified polysaccharide to the synthetic polymer is (0.5-5): (5-9.5); further, (1-3): (7-9).
Preferably, the preparation method of the anisotropic nanofiber comprises the following steps: mixing the modified polysaccharide with a synthetic polymer to obtain a mixture; preparing spinning solution by taking the mixture as solute; and then carrying out electrostatic spinning to obtain the anisotropic nanofiber.
Preferably, the solvent of the spinning solution is a mixture of hexafluoroisopropanol and acetic acid.
Preferably, the mass ratio of the hexafluoroisopropanol to the acetic acid is (8-10): 1, a step of; further 9:1.
preferably, the concentration of the acetic acid is 80-95 v/v%; further 90%.
Preferably, the electrostatic spinning adopts a roller receiving method, namely a roller receiver is used as a receiving device.
Preferably, the concentration of the mixture in the spinning solution is 0.5 to 15wt%; further 1 to 10%.
Preferably, the step of electrospinning is as follows: the spinning solution is put into an injection device, the injection device is pushed at a pushing speed of 0.1-2 mL/h, the distance between the needle head of the injection device and a receiving device is 10-20 cm, the rotating speed of the receiving device is 500-3500 rpm, and the spinning voltage is 10-30 kV.
Preferably, the propelling speed is 0.4-1 mL/h.
Preferably, the inner diameter of the needle of the injection device is 0.3-1.0 mm; further 0.5 to 1.0mm.
Preferably, the needle of the injection device is located at a distance of 12-15 cm from the receiving device.
Preferably, the method comprises the steps of. The needle head is connected with the anode, and the receiving device is connected with the cathode.
Preferably, the receiving device is coated with tinfoil.
Preferably, the rotation speed of the receiving device is 1000-3000 rpm.
Preferably, the spinning voltage is 20-22.5 kV.
In a second aspect of the present invention, there is provided a method for preparing the anisotropic nanofiber composite polysaccharide hydrogel of the first aspect of the present invention, wherein the preparation method is S11 or S12:
s11: mixing the modified polysaccharide with a photoinitiator to obtain a mixture A; laminating the mixture A with anisotropic nanofiber, and photo-curing to obtain an anisotropic nanofiber composite polysaccharide hydrogel containing an S1 anisotropic nanofiber layer and an S2 modified polysaccharide layer;
S12: mixing the functional substance, the photoinitiator and the modified polysaccharide to obtain a mixture B; and laminating the mixture B with the anisotropic nanofiber, and photo-curing to obtain the anisotropic nanofiber composite polysaccharide hydrogel containing the S1 anisotropic nanofiber layer and the S2 modified polysaccharide layer containing the functional substance.
Preferably, the preparation method is S11 or S12:
s11: mixing the modified polysaccharide with a photoinitiator to obtain a mixture A; alternately laminating the mixture A and the anisotropic nanofiber, and photo-curing to obtain the anisotropic nanofiber composite polysaccharide hydrogel;
s12: mixing the functional substance, the photoinitiator and the modified polysaccharide to obtain a mixture B; and alternately laminating the mixture B and the anisotropic nanofiber, and photo-curing to obtain the anisotropic nanofiber composite polysaccharide hydrogel.
Preferably, the concentration of the modified polysaccharide in S11 is 0.5-18% by mass-volume ratio; further 1 to 15.05 percent.
Preferably, the solvent of the modified polysaccharide in S11 is water.
Preferably, the photoinitiator comprises an Irgacure series photoinitiator.
Preferably, the final concentration of the photoinitiator in the mixture A or the mixture B is 0.01-0.1% by mass volume ratio; further 0.03 to 0.06 percent; further 0.03 to 0.05%.
Preferably, the concentration of the modified polysaccharide in the S12 is 8-16% in terms of mass-volume ratio; further 10 to 15.05 percent.
Preferably, the solvent of the modified polysaccharide in S12 is a culture medium; further DMEM-F12 medium.
Preferably, when the functional substance in S12 is stem cells, the ratio of the functional substance to the mixture of the modified polysaccharide and the photoinitiator is 10 5 ～10 8 cell/mL mixture; further 10 6 ～10 7 cell/mL mixture.
Preferably, the method of photocuring is S111 and/or S112:
s111: photo-curing under ultraviolet light;
s112: realized by a 3D printer.
Preferably, S111 is: photo-curing for 1-10 min under 360-370 nm ultraviolet light; the method further comprises the following steps: photo-curing for 5min under 365nm ultraviolet light.
Preferably, the 3D printer is a 3D printer adopting a stereoscopic light curing forming method (SLA) working principle, and is preferably a 3D printer with a model number of 3D systems ProX 800.
In a third aspect of the invention, there is provided the use of the anisotropic nanofiber composite polysaccharide hydrogel of the first aspect in the preparation of biomedical materials.
Preferably, the biomedical material is used for tissue repair; further preferably, the biomedical material is used for soft tissue repair; still further preferably, the biomedical material is used for heart valve tissue repair.
In a fourth aspect of the invention, there is provided a biomedical material comprising the anisotropic nanofiber composite polysaccharide hydrogel of the first aspect of the invention.
Preferably, the biomedical material is used for tissue repair; further preferably, the biomedical material is used for soft tissue repair; still further preferably, the biomedical material is used for heart valve tissue repair.
The beneficial effects of the invention are as follows:
the invention provides an anisotropic nanofiber composite polysaccharide hydrogel, which comprises the following components in parts by weight: s1: an anisotropic nanofiber layer, the anisotropic nanofiber being prepared from the following raw materials: modified polysaccharide and synthetic polymer; s2: a modified polysaccharide layer; or a modified polysaccharide layer comprising a functional substance; the hydrogel has photocuring capability and printability through modified polysaccharide; the hydrogel has good mechanical properties through the anisotropic nanofiber, and simultaneously, the osteogenic differentiation of cells is inhibited; the hydrogel can be used for preparing biomedical materials with tissue (such as vascular prongs, heart valves and the like) repairing functions.
Drawings
Fig. 1 is a graph of strain energy data (n=3) for cell-free hydrogels prepared in comparative examples 1 to 3 and examples 1 to 4.
FIG. 2 is a graph showing the expression levels of the Runx2 gene and osteocalcin OCN gene in HADMSC cells in the cell-carrying hydrogels of examples 6 to 9 and comparative examples 4 to 6 (n=3).
Detailed Description
The present invention will be described in further detail with reference to specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and the like used in this example are commercially available ones unless otherwise specified.
Example 1 preparation method of Anisotropic nanofiber composite Natural polysaccharide hydrogel
(1) Preparation of modified gelatin: 10G of gelatin (Sigma Co., G9382, from cow leather, number average molecular weight (Mn) of 80000D) was weighed and dissolved in 100mL of ultrapure water to prepare a gelatin solution having a mass to volume ratio (G/mL) of 10%, and then pH was adjusted to 8.5 with a 5mol/L NaOH solution; adding 7.5mL of methacrylic anhydride dropwise, and reacting for 6 hours at 40 ℃, wherein the pH value of the reaction solution is controlled to be 8-9 through NaOH in the whole reaction process; and (3) placing the product obtained by the reaction in a dialysis bag with the molecular weight cut-off of 3500, dialyzing for 3 days, and freeze-drying to obtain the modified gelatin.
(2) Preparation of anisotropic nanofibers: taking polycaprolactone (PCL, aba Ding Gongsi, CAS number: 24980-41-4; mn=60000-65000D) and the modified gelatin obtained in the step (1) (mass ratio of 7:3) to obtain a mixture, dissolving the mixture in a mixed solvent system of Hexafluoroisopropanol (HFIP) and 90v/v% acetic acid (mass ratio of HFIP to 90% acetic acid is 9:1), preparing a spinning solution, wherein the concentration of the mixture in the spinning solution is 10wt%, sucking the spinning solution into a 2.5mL syringe (the pushing speed of the syringe is 0.5 mL/h), the inner diameter of a metal needle of the syringe is 0.5mm, and the distance from the cylinder support is 15cm, and preparing a tubular support consisting of composite nanofibers by exchanging for a receiving device with different diameters, wherein the diameter of the cylinder is set to be 2.0cm, and the rotating speed of the roller is 2000rpm; under the action of high voltage (22.5 kV), the spinning solution is sprayed in an electric field to form fibers which are deposited on a roller receiver to form a fiber film; the electrostatic spinning voltage and the spinning speed are randomly regulated according to the spinning condition, and when the required thickness is reached (0.3 mm in this example), the aluminum foil and the fiber film are carefully peeled off from the surface of the roller; observing the morphology of the electrospun fibers according to a Scanning Electron Microscope (SEM) and taking a photograph; based on the obtained SEM Image, the electrospun fiber scanning electron microscope Image is converted into a 2D-FFT Image of gray level Image (8-bit, 2048x2048 pixels), and then the Image analysis software Image J and plug-in value profile thereof are processed, and the prepared nanofiber orientation degree is analyzed and calculated, wherein the orientation degree is 0.118.
(3) Preparation of composite natural polysaccharide hydrogel: preparing 50mL of modified gelatin solution A with the concentration of 10% by mass and volume by using ultrapure water, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified gelatin solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass and volume; and (3) compounding the solution C with the anisotropic nanofiber membrane prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting the nanofiber membrane into the disc die, wherein the number of nanofiber layers is 3, and the upper layer and the lower layer are the solution C), and then placing the disc die under 365nm ultraviolet light for photocuring for 5min (when a specific shape is needed, the solution C can be printed and molded through 3D systems ProX 800, and photocuring is carried out in the printing process), so that the anisotropic nanofiber composite natural polysaccharide hydrogel is prepared.
Example 2 preparation method of Anisotropic nanofiber composite Natural polysaccharide hydrogel
(1) Preparation of modified hyaluronic acid: 1g of hyaluronic acid (Sigma Co., CAS No.:9067-32-7, mn=1200000D) was weighed and dissolved in 100mL of ultrapure water to prepare a hyaluronic acid solution having a mass/volume ratio of 1%, and then the pH was adjusted to 8.5 with a NaOH solution having a concentration of 5 mol/L; adding 7.5mL of methacrylic anhydride dropwise, and reacting for 6 hours at 40 ℃, wherein the pH value of the reaction solution is controlled to be 8-9 through NaOH in the whole reaction process; and (3) placing the product obtained by the reaction in a dialysis bag with the molecular weight cut-off of 3500, dialyzing for 3 days, and freeze-drying to obtain the modified hyaluronic acid.
(2) Preparation of anisotropic nanofibers: taking polyethylene glycol (Aba Ding Gongsi, CAS number: 25322-68-3, mn=8000) and modified hyaluronic acid obtained in the step (1) (the mass ratio is 8:2), obtaining a mixture, dissolving the mixture in a mixed solvent system of Hexafluoroisopropanol (HFIP) and 90v/v% acetic acid (the mass ratio of HFIP to 90% acetic acid is 9:1), preparing a spinning solution, wherein the concentration of the mixture in the spinning solution is 1wt%, sucking the spinning solution into a 2.5mL syringe (the pushing speed of the syringe is 0.5 mL/h), the inner diameter of a metal needle of the syringe is 0.5mm, and the distance from a roller bracket is 12cm, and preparing a tubular bracket consisting of composite nano fibers by exchanging a receiving device with different diameters, wherein the diameter of the roller is set to be 2.0cm, and the rotating speed of the roller is 3000rpm; under the action of high voltage (22.5 kV), the spinning solution is sprayed in an electric field to form fibers which are deposited on a roller receiver to form a fiber film; the electrostatic spinning voltage and the spinning speed are randomly regulated according to the spinning condition, and when the required thickness is reached (1 mm in this example), the aluminum foil and the fiber film are carefully peeled off from the surface of the roller; observing the morphology of the electrospun fibers according to a Scanning Electron Microscope (SEM) and taking a photograph; based on the obtained SEM Image, the electrospun fiber scanning electron microscope Image is converted into a 2D-FFT Image of gray level Image (8-bit, 2048x2048 pixels), and then the Image analysis software Image J and plug-in value profile thereof are processed, and the prepared nanofiber orientation degree is analyzed and calculated, wherein the orientation degree is 0.116.
(3) Preparation of composite natural polysaccharide hydrogel: preparing 50mL of modified hyaluronic acid solution A with the concentration of 1% by mass and volume by using ultrapure water, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified hyaluronic acid solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass and volume; and (3) compounding the solution C with the anisotropic nanofiber prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting nanofibers with the number of nanofiber layers of 3 and the upper and lower layers of the solution C), and then placing the solution C under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, printing and forming the solution C by 3D systems ProX 800 and photo-curing in the printing process), thereby obtaining the anisotropic nanofiber composite natural polysaccharide hydrogel.
Example 3 preparation method of Anisotropic nanofiber composite Natural polysaccharide hydrogel
(1) Preparation of modified sodium alginate: 1g of sodium alginate (Aba Ding Gongsi, CAS number 9005-38-3, mn=60000D) is weighed and dissolved in 100mL of ultrapure water to prepare sodium alginate solution with the mass-volume ratio of 1%, and then the pH value is adjusted to 8.5 by using NaOH solution with the concentration of 5 mol/L; adding 7.5mL of methacrylic anhydride dropwise, and reacting for 6 hours at 40 ℃, wherein the pH value of the reaction solution is controlled to be 8-9 through NaOH in the whole reaction process; and (3) placing the product obtained by the reaction in a dialysis bag with the molecular weight cut-off of 3500, dialyzing for 3 days, and freeze-drying to obtain the modified sodium alginate.
(2) Preparation of anisotropic nanofibers: taking polylactic acid (Aba Ding Gongsi, CAS number: 26100-51-6, mn=60000) and modified sodium alginate (mass ratio is 9:1) obtained in the step (1), obtaining a mixture, dissolving the mixture in a mixed solvent system of Hexafluoroisopropanol (HFIP) and 90v/v% acetic acid (mass ratio of HFIP to 90% acetic acid is 9:1), preparing a spinning solution, wherein the concentration of the mixture in the spinning solution is 1wt%, sucking the spinning solution into a 2.5mL syringe (the pushing speed of the syringe is 0.5 mL/h), the inner diameter of a metal needle of the syringe is 0.5mm, and the distance from the cylinder support is 14cm, preparing a tubular support consisting of composite nanofibers by exchanging a receiving device with different diameters, wherein the diameter of the cylinder is set to be 2.0cm, and the rotating speed of the roller is 1000rpm; under the action of high voltage (22.5 kV), the spinning solution is sprayed in an electric field to form fibers which are deposited on a roller receiver to form a fiber film; the electrostatic spinning voltage and the spinning speed are randomly regulated according to the spinning condition, and when the required thickness is reached (1 mm in this example), the aluminum foil and the fiber film are carefully peeled off from the surface of the roller; observing the morphology of the electrospun fibers according to a Scanning Electron Microscope (SEM) and taking a photograph; based on the obtained SEM Image, the electrospun fiber scanning electron microscope Image is converted into a 2D-FFT Image of a gray level Image (8-bit, 2048x2048 pixels), and then the Image analysis software Image J and an plug-in value profile thereof are processed, and the prepared nanofiber orientation degree is analyzed and calculated, wherein the orientation degree is 0.113.
(3) Preparation of composite natural polysaccharide hydrogel: preparing 50mL of modified sodium alginate solution A with the concentration of 1% by mass and volume of the modified sodium alginate prepared in the step (1) by using ultrapure water, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified sodium alginate solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass and volume; and (3) compounding the solution C with the anisotropic nanofiber prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting nanofibers with the number of nanofiber layers of 2 and the upper and lower layers of the solution C), and then placing the solution C under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, printing and forming the solution C by 3D systems ProX 800 and photo-curing in the printing process), thereby obtaining the anisotropic nanofiber composite natural polysaccharide hydrogel.
Example 4 preparation method of Anisotropic nanofiber composite Natural polysaccharide hydrogel
(1) Preparation of modified gelatin and modified sodium alginate: the preparation methods of the modified gelatin and the modified sodium alginate are the same as those of examples 1 and 3.
(2) Preparation of anisotropic nanofibers: the anisotropic nanofibers were prepared in the same manner as in example 1 (except that modified gelatin was replaced with a mixture of modified gelatin and modified sodium alginate, wherein the mass ratio of modified gelatin to modified sodium alginate was 10:1), and the degree of orientation was 0.118.
(3) Preparation of composite natural polysaccharide hydrogel: preparing modified gelatin solution with the concentration of 20% by mass and volume and modified sodium alginate solution with the concentration of 2% by mass and volume respectively from the modified gelatin and the modified sodium alginate prepared in the step (1) by using ultrapure water; mixing 20wt% of modified gelatin and 2wt% of modified sodium alginate according to a volume ratio of 1:1 to prepare a modified natural polysaccharide hydrogel solution A50mL, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified natural polysaccharide hydrogel solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio; and (3) compounding the solution C with the anisotropic nanofiber prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting nanofibers with the number of nanofiber layers of 3 and the upper and lower layers of the solution C), and then placing the solution C under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, printing and forming the solution C by 3D systems ProX 800 and photo-curing in the printing process), thereby obtaining the anisotropic nanofiber composite natural polysaccharide hydrogel.
Example 5 preparation method of Anisotropic nanofiber composite Natural polysaccharide hydrogel
(1) Preparation of modified gelatin and modified hyaluronic acid: the preparation methods of the modified gelatin and the modified hyaluronic acid are the same as in examples 1 and 2.
(2) Preparation of anisotropic nanofibers: the anisotropic nanofibers were prepared in the same manner as in example 2 (except that modified hyaluronic acid was replaced with a mixture of modified gelatin and modified hyaluronic acid, wherein the mass ratio of modified gelatin to modified hyaluronic acid was 30:1), and the degree of orientation was 0.115.
(3) Preparation of composite natural polysaccharide hydrogel: preparing modified gelatin solution with the concentration of 20% by mass and volume ratio and modified hyaluronic acid solution with the concentration of 2% by mass and volume ratio respectively from the modified gelatin and the modified hyaluronic acid prepared in the step (1) by using ultrapure water; mixing 20% modified gelatin and 2% modified hyaluronic acid according to a volume ratio of 3:1 to prepare a modified natural polysaccharide hydrogel solution A50 mL, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified natural polysaccharide hydrogel solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio; and (3) compounding the solution C with the anisotropic nanofiber prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting nanofibers with the number of nanofiber layers of 3 and the upper and lower layers of the solution C), and then placing the solution C under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, printing and forming the solution C by 3D systems ProX 800 and photo-curing in the printing process), thereby obtaining the anisotropic nanofiber composite natural polysaccharide hydrogel.
Example 6 preparation method of cell-loaded Anisotropic nanofiber composite Natural polysaccharide hydrogel
The preparation method of this example is the same as that of example 1, except that step (3) is:
preparation of cell-carrying composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, dissolving the modified gelatin prepared in the step (1) in a DMEM-F12 culture medium, wherein the concentration of the modified gelatin in the obtained solution B is 10% by mass and volume; adding a solution of a photoinitiator Irgacure2959 (CIBA Chemicals) to the solution B to obtain a solution D, wherein the final concentration of the Irgacure2959 is 0.05% by mass volume; press 10 7 cell/mL amount of solution D, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution D to obtain solution E; injecting the solution E into the upper layer and the lower layer of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively with the thickness of 1mm, adding the 0.3mm anisotropic nanofiber prepared in the step (2) in the middle, and then placing the disc sheet die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, the solution E can be printed and molded through 3D systems ProX 800, and photo-curing is carried out in the printing process), thus obtaining the cell-carrying composite natural polysaccharide hydrogel.
Example 7 preparation method of cell-loaded Anisotropic nanofiber composite Natural polysaccharide hydrogel
The preparation method of this example is the same as that of example 2, except that step (3) is:
preparation of cell-carrying composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, dissolving the modified hyaluronic acid prepared in the step (1) in a DMEM-F12 culture medium, wherein the concentration of the modified hyaluronic acid in the obtained solution B is 1% by mass and volume; adding a solution of a photoinitiator Irgacure 2959 (CIBA Chemicals) to the solution B to obtain a solution D, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume; press 10 7 cell/mL amount of solution D, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution D to obtain solution E; injecting the solution E into the upper layer and the lower layer of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively for 1mm, adding the 1mm anisotropic nanofiber prepared in the step (2) in the middle, and then placing the disc sheet die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, the solution E can be printed and molded through 3D systems ProX 800, and photo-curing is carried out in the printing process), thus obtaining the cell-carrying composite natural polysaccharide hydrogel.
Example 8 preparation method of cell-loaded Anisotropic nanofiber composite Natural polysaccharide hydrogel
The preparation method of this example is the same as that of example 3, except that step (3) is:
preparation of cell-carrying composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, and preparing the material in the step (1)The modified sodium alginate is dissolved in a DMEM-F12 culture medium, and the concentration of the modified sodium alginate in the obtained solution B is 1% by mass and volume; adding a solution of a photoinitiator Irgacure 2959 (CIBA Chemicals) to the solution B to obtain a solution D, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume; press 10 7 cell/mL amount of solution D, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution D to obtain solution E; injecting the solution E into the upper layer and the lower layer of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively for 1mm, adding the 1mm anisotropic nanofiber prepared in the step (2) in the middle, and then placing the disc sheet die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, the solution E can be printed and molded through 3D systems ProX 800, and photo-curing is carried out in the printing process), thus obtaining the cell-carrying composite natural polysaccharide hydrogel.
Example 9 preparation method of cell-loaded Anisotropic nanofiber composite Natural polysaccharide hydrogel
The preparation method of this example is the same as that of example 4, except that step (3) is:
preparation of cell-carrying composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, and respectively dissolving the modified gelatin and the modified sodium alginate prepared in the step (1) in a DMEM-F12 culture medium to obtain a modified gelatin solution with the concentration of 20% by mass and volume and a modified sodium alginate solution with the mass and volume ratio of 2%; mixing 20% of modified gelatin and 2% of modified sodium alginate according to a volume ratio of 1:1 to obtain a solution B, wherein the concentration of the modified sodium alginate in the solution B is 1% by mass and volume ratio, and the concentration of the modified gelatin is 10% by mass and volume ratio; adding a solution of a photoinitiator Irgacure 2959 (CIBA Chemicals) to the solution B to obtain a solution D, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume; press 10 7 cell/mL amount of solution D, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution D to obtain solution E; injecting the solution E into the upper and lower layers of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively with the thickness of 1mm, adding the 0.3mm anisotropic nanofiber prepared in the step (2) in the middle, and then placing the disc sheet die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, the solution E can be printed and molded through 3D systems ProX 800, and photo-curing is carried out in the printing process), so that the cell-carrying composite natural polysaccharide hydrogel is prepared.
Example 10 preparation method of cell-loaded Anisotropic nanofiber composite Natural polysaccharide hydrogel
The preparation method of this example is the same as that of example 5, except that step (3) is:
preparation of cell-carrying composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, and respectively dissolving the modified gelatin and the modified hyaluronic acid prepared in the step (1) in a DMEM-F12 culture medium to obtain a modified gelatin solution with the concentration of 20% by mass and volume and a modified hyaluronic acid solution with the mass and volume ratio of 2%; mixing 20% of modified gelatin and 2% of modified hyaluronic acid according to a volume ratio of 3:1 to obtain a solution B, wherein the concentration of the modified hyaluronic acid in the solution B is 0.05% by mass and volume ratio, and the concentration of the modified gelatin is 15% by mass and volume ratio; adding a solution of a photoinitiator Irgacure 2959 (CIBA Chemicals) to the solution B to obtain a solution D, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume; press 10 7 cell/mL amount of solution D, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution D to obtain solution E; injecting the solution E into the upper layer and the lower layer of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively for 1mm, adding the 1mm anisotropic nanofiber prepared in the step (2) in the middle, and then placing the disc sheet die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, the solution E can be printed and molded through 3D systems ProX 800, and photo-curing is carried out in the printing process), thus obtaining the cell-carrying composite natural polysaccharide hydrogel.
Comparative example 1 preparation method of hydrogel
(1) Preparation of modified gelatin: the preparation method of the modified gelatin is the same as that of example 1.
(2) Preparation of hydrogels: the modified gelatin is dissolved in deionized water to prepare a modified gelatin solution with the mass volume ratio of 10%, and then a photoinitiator Irgacure 2959 (CIBA Chemicals) solution is added, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio. Filling the modified gelatin solution added with the photoinitiator into a disc die with the diameter of 20mm and the thickness of 5mm, and then placing the disc die under 365nm ultraviolet light for photo-curing for 5min to obtain the hydrogel.
Comparative example 2 preparation method of random-oriented nanofiber composite natural polysaccharide hydrogel
(1) Preparation of modified gelatin: the modified gelatin was prepared in the same manner as in example 1.
(2) Preparation of randomly oriented (isotropic) nanofibers: taking Polycaprolactone (PCL) and modified gelatin obtained in the step (1) (the mass ratio is 7:3) to obtain a mixture, dissolving the mixture in a mixed solvent system of Hexafluoroisopropanol (HFIP) and 90v/v% acetic acid (the mass ratio of HFIP to 90% acetic acid is 9:1), preparing a spinning solution, wherein the concentration of the mixture in the spinning solution is 10wt%, and sucking the spinning solution into a 2.5mL syringe (the pushing speed of the syringe is 0.5 mL/h), and the inner diameter of a metal needle of the syringe is 0.5mm and is 15cm away from a receiving plate; under the action of high voltage (22.5 kV), the spinning solution is sprayed in an electric field to form fibers which are deposited on a flat plate receiver to form a fiber film; when the desired thickness is reached (0.3 mm in this example), the aluminum foil and fibrous film are carefully peeled off the receiving plate surface; based on the obtained SEM Image, the degree of orientation was 0.108, calculated according to the Image analysis software Image J process.
(3) Preparation of composite natural polysaccharide hydrogel: preparing 50mL of modified gelatin solution A with the concentration of 10% by mass and volume by using ultrapure water, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified gelatin solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass and volume; and (3) compounding the solution C with the random orientation nanofiber prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting the nanofiber layers of 3 and the upper and lower layers of the nanofiber into the disc die), and then placing the disc die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, printing and forming the solution C through 3D systems ProX 800 and photo-curing in the printing process) to obtain the random orientation nanofiber composite natural polysaccharide hydrogel.
Comparative example 3 preparation method of random-oriented nanofiber composite natural polysaccharide hydrogel
(1) Preparation of modified gelatin and modified sodium alginate: the preparation methods of the modified gelatin and the modified sodium alginate are the same as in example 4.
(2) Preparation of randomly oriented nanofibers: taking Polycaprolactone (PCL) and a mixture of modified gelatin and modified sodium alginate (the mass ratio of PCL to the mixture is 7:3, wherein the mass ratio of the modified gelatin to the modified sodium alginate in the mixture is 10:1) to obtain a mixture A, dissolving the mixture A in a mixed solvent system of Hexafluoroisopropanol (HFIP) and 90v/v% acetic acid (the mass ratio of HFIP to 90% acetic acid is 9:1), preparing a spinning solution, wherein the concentration of the mixture A in the spinning solution is 10wt%, sucking the spinning solution into a 2.5mL syringe (the pushing speed of the syringe is 0.5 mL/h), and the inner diameter of a metal needle of the syringe is 0.5mm and is 15cm away from a receiving plate; under the action of high voltage (22.5 kV), the spinning solution is sprayed in an electric field to form fibers which are deposited on a flat plate receiver to form a fiber film; when the desired thickness is reached (0.3 mm in this example), the aluminum foil and fibrous film are carefully peeled off the receiving plate surface; based on the obtained SEM Image, the degree of orientation was 0.108, calculated according to the Image analysis software Image J process.
(3) Preparation of composite natural polysaccharide hydrogel: preparing modified gelatin solution with the concentration of 20% by mass and volume and modified sodium alginate solution with the concentration of 2% by mass and volume respectively from the modified gelatin and the modified sodium alginate prepared in the step (1) by using ultrapure water; mixing 20% modified gelatin and 2% modified sodium alginate according to a volume ratio of 1:1 to prepare a modified natural polysaccharide hydrogel solution A50mL, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified natural polysaccharide hydrogel solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio; and (3) compounding the solution C with the random orientation nanofiber prepared in the step (2) (filling the solution C into a disc die with the diameter of 20mm and the thickness of 5mm, alternately putting the nanofiber layers of 3 and the upper and lower layers of the nanofiber into the disc die), and then placing the disc die under 365nm ultraviolet light for photo-curing for 5min (when a specific shape is needed, printing and forming the solution C through 3D systems ProX 800 and photo-curing in the printing process) to obtain the random orientation nanofiber composite natural polysaccharide hydrogel.
Comparative example 4 preparation of cell-carrying hydrogel
The preparation method of this comparative example is the same as that of comparative example 1, except that step (2) is:
(2) Preparation of cell-carrying hydrogels:
sterilizing all materials for more than 1h under ultraviolet, dissolving the modified gelatin solution in a DMEM-F12 culture medium according to a mass volume ratio of 10%, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) solution to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio. Press 10 7 cell/mL amount of solution C, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution C to obtain solution E. And (3) injecting the solution E into a disc die with the diameter of 20mm and the thickness of 5mm, and then placing the disc die under 365nm ultraviolet light for photo-curing for 5min to obtain the cell-carrying hydrogel.
Comparative example 5 preparation method of cell-loaded random-oriented nanofiber composite natural polysaccharide hydrogel
The preparation method of this comparative example is the same as that of comparative example 2, except that step (3) is:
(3) Preparation of cell-carrying random orientation nanofiber composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, dissolving the modified gelatin prepared in the step (1) in a DMEM-F12 culture medium according to a mass volume ratio of 10%, and adding a solution of a photoinitiator Irgacure 2959 (CIBA Chemicals company) to obtain a solution D, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio. Press 10 7 cell/mL amount of solution D, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma Co.) were inoculated into solution D to obtain solution E. Injecting the solution E into the upper layer and the lower layer of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively with the thickness of 1mm, adding the 0.3mm random orientation nanofiber prepared in the step (2) in the middle, and then placing the mixture in 365nm ultraviolet light for photo-curing for 5min to obtain the cell-carrying random orientation nanofiber composite natural polysaccharide hydrogel.
Comparative example 6 preparation method of cell-loaded random-oriented nanofiber composite natural polysaccharide hydrogel
The preparation method of this comparative example is the same as that of comparative example 3, except that step (3) is:
(3) Preparation of composite natural polysaccharide hydrogel: sterilizing all materials for more than 1h under ultraviolet, and respectively preparing modified gelatin solution with the concentration of 20% by mass and volume and modified sodium alginate solution with the concentration of 2% by mass and volume by using a DMEM-F12 culture medium; mixing 20% of modified gelatin and 2% of modified sodium alginate according to a volume ratio of 1:1 to prepare a modified natural polysaccharide hydrogel solution A, and adding a photoinitiator Irgacure 2959 (CIBA Chemicals company) into the modified natural polysaccharide hydrogel solution A to obtain a solution C, wherein the final concentration of the Irgacure 2959 is 0.05% by mass volume ratio; press 10 7 cell/mL amount of solution C, human adipose-derived mesenchymal stem cells (HADMSC cells, sigma) were inoculated into solution C to obtain solution E; injecting the solution E into the upper layer and the lower layer of a disc sheet die with the diameter of 8mm and the thickness of 3mm respectively with the thickness of 1mm, adding the 0.3mm random orientation nanofiber prepared in the step (2) in the middle, and then placing the mixture in 365nm ultraviolet light for photo-curing for 5min to obtain the cell-carrying random orientation nanofiber composite natural polysaccharide hydrogel.
Effect examples
(1) The cell-free hydrogels prepared in comparative examples 1 to 3 and examples 1 to 4 were subjected to stress strain test by a compression model using a dynamic thermo-mechanical analyzer, and then the formula σ=ce was calculated from the formula strain energy αε And calculating to obtain the strain energy. The strain energy calculation results are shown in fig. 1: the strain energy of examples 1-4 is much higher than that of comparative examples 1-3; the addition of nanofibers was shown to increase the strain energy (mechanical properties) of the hydrogels, where the strain energy promoted by the addition of anisotropic nanofibers is significantly higher than that of randomly oriented nanofibers.
(2) After the cell-carrying hydrogels of examples 6 to 9 and comparative examples 4 to 6 were cultured in DMEM F12 medium for 14 days, runx2 gene and osteocalcin OCN gene were detected by qPCR as follows:
(1) And (3) separating and purifying RNA: taking one hydrogel disc (with the diameter of 8mm and the thickness of 3 mm) after culturing for 14 days, adding 0.5mL of TRIzol, mashing, standing for 5min, adding 0.1mL of chloroform, standing for 2min after uniform mixing, centrifuging for 15min at the temperature of 12000rcf at the temperature of 4 ℃, transferring upper-layer aqueous-phase RNA to a new centrifuge tube, adding 0.5mL of isopropanol containing glycogen blue into the centrifuge tube, standing for 10min after mixing, centrifuging for 10min at the temperature of 10000rcf at the temperature of 4 ℃, removing supernatant, adding 0.7mL of 75% ethanol, centrifuging for 5min at the temperature of 7500rcf at the temperature of 4 ℃ after uniform mixing, removing supernatant, adding 75% ethanol at the temperature of 7500rcf at the temperature of 4 ℃ after uniform mixing, adding 20 microliter of nuclease-free water after naturally airing for 20min, beating, and detecting the purity by using Nanodrop 2000;
(2) synthesis of cDNA: preparing a reaction solution containing qScript, adding the extracted RNA, and then completing cDNA synthesis and cloning on Eppendorf Mastercycler equipment;
(3) qPCR: preparing an upstream primer and a downstream primer containing 18s, runx2 and OCN genes, adding the cDNA obtained in the step (2) into the reaction solution of SYBR, centrifuging for 10s after membrane sealing, and completing qPCR in Bio-rad CFX connect Real time system, wherein the adopted PCR reaction system is as follows:
6. Mu.L of nuclease-free water, 1. Mu. L, SYBR 10. Mu.L of each of the 10. Mu.M concentration of the upstream primer and the 10. Mu.M concentration of the downstream primer, and 2. Mu.L of the template cDNA (step (2) synthesis).
The PCR reaction conditions were: denaturation at 95℃for 5min;94 ℃ 45s, 55 ℃ 45s, 72 ℃ 60s,30 cycles; extending at 72℃for 10min.
18s upstream primer: 5'-CCAACCTGGTTGATCCTGCCAGTA-3' (SEQ ID NO. 1);
18s downstream primer: 5'-CCTTGTTAACGACTTCACCTTCCTCT-3' (SEQ ID NO. 2);
OCN upstream primer: 5'-GGCAGCGAGGTAGTGAAGA-3' (SEQ ID NO. 3);
OCN downstream primer: 5'-CCTGAAAGCCGATGTGGT-3' (SEQ ID NO. 4);
runx2 upstream primer: 5'-CCTCCTACCTGAGCCAGATG-3' (SEQ ID NO. 5);
runx2 downstream primer: 5'-CCAGAGGCAGAAGTCAGAGG-3' (SEQ ID NO. 6).
The results are shown in FIG. 2: compared to the comparative group (comparative examples 4 to 6), the expression amounts of Runx2 gene and osteocalcin OCN gene of the example group (examples 6 to 9) were significantly reduced after 14 days of culture, indicating that: the hydrogel provided by the invention is favorable for inhibiting osteogenic differentiation, namely, the osteogenic differentiation can be inhibited by adding the nanofiber, wherein the effect of inhibiting the osteogenic differentiation by adding the anisotropic nanofiber is obviously higher than that of inhibiting the osteogenic differentiation by adding the random orientation nanofiber.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An anisotropic nanofiber composite polysaccharide hydrogel comprising S1 and S2:
s1: an anisotropic nanofiber layer, the anisotropic nanofiber being prepared from the following raw materials: modified polysaccharide and synthetic polymer;
s2: a modified polysaccharide layer; or (b)
A modified polysaccharide layer comprising a functional material;
the modified polysaccharide comprises at least one of a methacryloyl group modified polysaccharide and a methacryloxypropyl group modified polysaccharide;
the synthetic polymer comprises at least one of polycaprolactone, polylactic acid, polyethylene glycol, polybutylene terephthalate-adipate and polyglycolide;
the functional substance comprises at least one of a cell growth factor and a stem cell.
2. The hydrogel of claim 1, wherein:
the orientation degree of the anisotropic nanofiber is 0.112-0.125; further 0.113 to 0.118;
Preferably, the thickness of the anisotropic nanofiber is 0.1 to 1.3mm.
3. The hydrogel according to claim 1 or 2, characterized in that:
the preparation method of the anisotropic nanofiber comprises the following steps: mixing the modified polysaccharide with a synthetic polymer to obtain a mixture; preparing spinning solution by taking the mixture as solute; and then carrying out electrostatic spinning to obtain the anisotropic nanofiber.
4. The hydrogel of claim 3, wherein:
the electrostatic spinning adopts a roller receiving method.
5. The hydrogel of claim 4, wherein:
the step of electrostatic spinning is as follows: the spinning solution is put into an injection device, the injection device is pushed at a pushing speed of 0.1-2 mL/h, the distance between the needle head of the injection device and a receiving device is 10-20 cm, the rotating speed of the receiving device is 500-3500 rpm, and the spinning voltage is 10-30 kV.
6. The hydrogel according to any one of claims 1 to 5, wherein:
the polysaccharide comprises at least one of gelatin, hyaluronic acid, sodium alginate, carrageenan and dextran;
preferably, the S1 and S2 are alternately laminated;
preferably, the number of layers of the S1 is n, and the number of layers of the S2 is n+1 or n-1;
Preferably, n is 2, 3, 4, 5, or 6.
7. The method for preparing the anisotropic nanofiber composite polysaccharide hydrogel according to any one of claims 1 to 6, which is characterized in that:
the preparation method is as follows:
s11: mixing the modified polysaccharide with a photoinitiator to obtain a mixture A; laminating the mixture A with anisotropic nanofiber, and photo-curing to obtain an anisotropic nanofiber composite polysaccharide hydrogel containing an S1 anisotropic nanofiber layer and an S2 modified polysaccharide layer;
s12: mixing the functional substance, the photoinitiator and the modified polysaccharide to obtain a mixture B; and laminating the mixture B with the anisotropic nanofiber, and photo-curing to obtain the anisotropic nanofiber composite polysaccharide hydrogel containing the S1 anisotropic nanofiber layer and the S2 modified polysaccharide layer containing the functional substance.
8. The method of manufacturing according to claim 7, wherein:
the photoinitiator comprises Irgacure series photoinitiators;
preferably, the method of photocuring is S111 and/or S112:
s111: photo-curing under ultraviolet light;
s112: realized by a 3D printer.
9. Use of an anisotropic nanofiber composite polysaccharide hydrogel according to any of claims 1 to 6 or a method of preparation according to any of claims 7 to 8 for the preparation of biomedical materials.
10. A biomedical material comprising the anisotropic nanofiber composite polysaccharide hydrogel of any one of claims 1-6.
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