CN113975460B - Bone repair scaffold material capable of mediating neurogenesis and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a bone repair scaffold material capable of mediating neurogenesis, a preparation method and application thereof. The bone repair scaffold material capable of mediating the nerve formation has good cell compatibility, osteogenic activity and calcium deposition promoting effect, can promote the proliferation, differentiation and mineralization process of mesenchymal stem cells towards the osteogenic direction, can also promote the survival process of neurons, and has wide prospect in clinical application of bone defect repair and restoration of perception of damaged parts as a bone repair material.
Description
Technical Field
The invention belongs to the technical field of biomedical composite materials, and particularly relates to a bone repair scaffold material capable of mediating neurogenesis, and a preparation method and application thereof.
Background
Bone is in accordance with the developmental stages of the nerve, which extends into bone development in a specific pattern, forming a "neuro-bone" structure. In mature bone, the bone is tightly bound to the nerves. Neuropeptides secreted by nerve fibers in bone tissue promote bone repair after bone injury, and few nerve fibers are found in callus in clinical cases of nonunion and delayed healing of bone defects. The result shows that the process of bone repair can be remarkably promoted by the neurogenesis of the bone defect part, and the neurogenesis of the bone defect part directly influences the recovery of the motor ability and the life quality of a patient and is an important reference for measuring the bone repair effect.
The main purpose of the nerve growth process is to promote the growth of nerves, and neurotrophic factors (NTF) can mediate the nerve growth process, such as Nerve Growth Factors (NGF) and brain-derived neurotrophic factors (BDNF) can promote the differentiation of nerve stem cells into neurons, promote the increase of the number of neurites of the neurons differentiated by the nerve stem cells, and have obvious effect of promoting the regeneration of the nerves. The existing research proves that NGF has potential nerve repair capability, can promote neurogenesis and angiogenesis of bone defects, can expand sensory nerve distribution so as to promote long bone development, and is important for primary and secondary ossification. In clinic, the NGF treatment continuously applied to the bone defect part can improve the bone repair rate and curative effect and enhance the remodeling activity of the bone in the cortex, and the addition of the NGF can achieve the effects of promoting the formation of new bone around the implant, promoting the maturation of trabecular bone, shortening the bone repair time and the like. BDNF has the functions of protecting bones and nerves, and promotes the osteogenesis and the neurogenesis of hBMSCs in vivo and in vitro, which suggests that BDNF can indirectly promote the osteogenesis by promoting the neurogenesis. Thus, promoting neurogenesis not only promotes sensory recovery, but can further promote the repair process of bone defects. Currently, there are related studies, for example, chinese patent application No. CN202010594528.1 discloses a bone repair scaffold material, a preparation method and an application thereof, the bone repair scaffold material is in a fibrous scaffold structure, and the bone repair scaffold material is composed of magnesium-doped mesoporous bioactive glass loaded with OOB fusion protein; the OOB fusion protein is formed by connecting OCN, OPN and BGN; the preparation method comprises the steps of preparing the magnesium-doped mesoporous bioactive glass, obtaining OOB fusion protein, loading and electrostatic spinning; the bone repair scaffold material has excellent bone repair performance and can provide long-acting supply for the bone repair process; the preparation method can successfully prepare the bone repair scaffold material containing the three proteins of OCN, OPN and BGN, and the bone repair scaffold material can be applied to clinical bone defect repair.
The bone matrix is in a nano fibrous grid structure, and provides a favorable structure for osteoblast proliferation, differentiation and bone matrix mineralization. The electrostatic spinning is carried out by spraying and spinning polymer solution or melt in a strong electric field, so that the nano-scale fiber grid support can be prepared, and the high fitting of the bone matrix structure is realized. The scaffold prepared by electrostatic spinning has a unique microstructure and proper mechanical properties, and can simulate a woven grid structure of a natural bone matrix to promote bone repair.
Therefore, the bone repair scaffold material prepared by the electrostatic spinning process can be fitted with a nano-fiber grid structure in a bone matrix, provides a suitable microenvironment for cells at a bone defect part, and improves a bone repair effect. The coaxial electrostatic spinning is used as a spinning system with double sources, so that various requirements can be integrated into one bracket material, and the preparation of the neurogenic bone defect repairing material is guaranteed.
Disclosure of Invention
The invention provides a bone repair scaffold material capable of mediating the neurogenesis, a preparation method and application thereof, aiming at the problems that in the prior art, a bone repair material only repairs bone defects and nerves are not repaired together, so that repaired parts do not feel, and the life quality of a patient is finally affected.
According to the preparation method of the bone repair scaffold material capable of mediating the neurogenesis, a magnesium-doped mesoporous bioactive glass (Mg-MBG) material is synthesized by a sol-gel and template method, OCN-OPN-BGN fusion protein (OOB fusion protein) is adsorbed in the Mg-MBG material and is used as a core of coaxial electrostatic spinning for spinning, so that long-acting supply can be provided for a bone repair process; the neurotrophic factor (NTF) is loaded in Silk Fibroin (SF) extracted by a boiling method, and is used as a shell of coaxial electrostatic spinning for spinning, so that support and supply can be provided for the neurogenesis in the bone repair process; therefore, the bone repair scaffold material with an osteogenic "core" and a neurogenic "shell" that can mediate neurogenesis provides a scaffold structure for osteogenesis and neurogenesis of a bone defect, and promotes the bone repair and neurogenesis process.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a bone repair scaffold material capable of mediating the neurogenesis comprises the following steps:
1) mixing and stirring OCN-OPN-BGN fusion protein (OOB fusion protein) heterologously expressed in vitro and magnesium-doped mesoporous bioactive glass (Mg-MBG) according to the proportion of (100) g: 300 Mg: 20Mg, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption, centrifuging at 12,000rpm for 5min, and taking the precipitate to obtain Mg-MBG material (OOB @ Mg-MBG) loaded with the OOB fusion protein;
2) according to the proportion of (3-8) g (2.2-4.4) g/L, enabling the silkworm cocoons to pass through Na 2 CO 3 Boiling the aqueous solution for degumming, drying, and obtaining dissolved silk fibroin through 7-10M lithium bromide solution;
dialyzing the dissolved silk fibroin by deionized water, and concentrating by 5-20% PEG solution with molecular weight of 10000;
finally centrifuging the concentrated silk fibroin for 2 times at 4 ℃, 7000-12000rpm for 20min each time, removing impurities, and finally obtaining a silk fibroin original solution;
3) mixing and stirring neurotrophic factor (NTF) and Silk Fibroin (SF) solution according to the proportion of (5-8) mug, (10-30) mg, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption, centrifuging the mixture at 12,000rpm for 5min, and taking the precipitate to obtain an NTF-loaded SF material (NTF @ SF);
4) placing poly-epsilon-caprolactone (PCL) in a hexafluoroisopropanol solution, and magnetically stirring according to the proportion of (4-6) g:50mL until the solution is completely clear to obtain a spinning solution;
adding the Mg-MBG material (OOB @ Mg-MBG) loaded with the OOB fusion protein obtained in the step 1) and the SF material (NTF @ SF) loaded with the NTF obtained in the step 3) into a spinning solution according to 5-15Mg/mL respectively, taking the Mg-MBG material (OOB @ Mg-MBG) loaded with the OOB fusion protein as an inner core and the SF material (NTF @ SF) loaded with the NTF as an outer shell, fully and uniformly mixing the solution through stirring and ultrasound, and performing electrostatic spinning under a high-voltage direct-current electric field of 18-22kV by using a digital injection pump at a speed of 0.60-0.85mL/h, at a temperature of 20-25 ℃ and a relative humidity of 30-60% to obtain the bone repair scaffold material capable of mediating the nerve.
In the invention: stirring in the step 1) for 1-2 h; the standing adsorption lasts for 24-48 h.
The drying in the step 2) is carried out by placing the mixture in a drying oven at the temperature of 40-80 ℃; the silk fibroin after being dissolved is obtained by dissolving the 7-10M lithium bromide solution in an oven at the temperature of 40-80 ℃ completely; the concentration is carried out for 12 to 60 hours.
Stirring for 1-2h in the step 3); the standing adsorption lasts for 24-48 h.
In the step 4), electrostatic spinning is carried out by a digital injection pump at a speed of 0.70mL/h, a temperature of 25 ℃ and a relative humidity of 44%.
The invention also relates to a bone repair scaffold material capable of mediating the neurogenesis, which is obtained by adopting the preparation method of the bone repair scaffold material capable of mediating the neurogenesis.
The bone repair scaffold material capable of mediating the neurogenesis can promote the osteogenesis process, and SF loaded with NTF can promote the neurogenesis process, so that the bone repair scaffold material has a wide application prospect in the bone defect repair process.
Compared with the prior art, the invention has the following advantages:
1. the invention relates to a preparation method of a bone repair scaffold material capable of mediating the nerve growth, which comprises the steps of adsorbing bone matrix fusion protein expressed in a heterologous way into mesoporous bioactive glass doped with calcium and magnesium prepared by a sol-gel and template self-assembly method to serve as an inner core, loading nerve growth factors for promoting the nerve growth into fibroin to serve as an outer shell, and preparing the bone repair scaffold material capable of mediating the nerve growth and having a core-shell structure by a coaxial electrostatic spinning technology. Compared with the existing material which only repairs bone tissues in bone repair, the invention has obvious advantages that the bone and nerves are repaired simultaneously, and the repaired nerves can further promote the repair of the bone tissues, thereby accelerating the rhythm of bone repair. Secondly, the existing material aims at repairing the vascularization of a bone tissue part in the bone repair process so as to promote the bone repair process, but the existing material cannot solve the problem of restoring consciousness of the repaired bone tissue part, and the bone repair scaffold material capable of mediating the vascularization can be used for double repair, namely bone repair and vascularization repair.
2. The bone repair scaffold material capable of mediating the neurogenesis, which is obtained by the invention, has good cell compatibility, osteogenic activity, neurogenic activity and the effect of promoting calcium deposition, can promote the proliferation, differentiation and mineralization process of mesenchymal stem cells in the osteogenic direction, can also promote the survival process of neurons, and has wide prospects in clinical application of bone defect repair and restoration of perception of damaged parts as a bone repair material.
3. The bone repair scaffold material capable of mediating the nerve transformation, which is obtained by the invention, has a nanofiber-shaped fiber structure, provides a microenvironment for the proliferation, differentiation and mineralization processes of cells, and realizes high fitting of a bone matrix structure, so that the bone repair process can be promoted, and nerve cells can be promoted to grow along nanofiber lines, thereby achieving the purpose of nerve repair.
Drawings
FIG. 1 is a microscopic surface structure of a bone repair scaffold material capable of mediating neurogenesis obtained in example 1 by scanning electron microscopy;
FIG. 2 is the Mg-MBG, OOB @ Mg-MBG and fluid simulating human body fluid (SBF) (142.0mM Na) of the bone repair scaffold capable of mediating neurogenesis of example 1 + ，5.0mM K + ，1.5mM Mg 2+ ，2.5mM Ca 2+ ，103.0mM Cl - ，27.0mM HCO 3- ，1.0mM HPO 4 2- ，0.5mM SO 4 2- ) The degree of mineralization in vitro is tested, and a scanning electron microscope image is adopted;
FIG. 3 is a graph of the proliferation potency of the Mg-MBG, OOB @ Mg-MBG and bone repair scaffold material mediating the neurogenesis in example 1 on bone marrow mesenchymal stem cells as determined by Cell Counting Kit-8 (CCK-8) reading absorbance values at a wavelength of 450nm with a microplate reader;
FIG. 4 is a graph in which the differentiation ability of Mg-MBG, OOB @ Mg-MBG and bone repair scaffold material mediating the neurogenesis in example 1 to mesenchymal stem cells is measured by measuring the activity of alkaline phosphatase, a prophase index of cell differentiation;
FIG. 5 is a graph of the mineralization capabilities of Mg-MBG, OOB @ Mg-MBG and bone repair scaffold materials mediating neurogenesis in example 1 on bone marrow mesenchymal stem cells as determined by alizarin red-stained mineralized nodules;
FIG. 6 is a graph of the ability of Mg-MBG, OOB @ Mg-MBG and bone repair scaffold materials that mediate neurogenesis in example 1 to differentiate bone marrow mesenchymal stem cells into the neural direction as measured by immunofluorescence.
Detailed Description
The present invention is described in further detail below by way of examples, which should not be construed as limiting the invention thereto. Example 1: a preparation method of a bone repair scaffold material capable of mediating the neurogenesis comprises the following steps:
1) mixing and stirring OCN-OPN-BGN fusion protein (OOB fusion protein) heterologously expressed in vitro and magnesium-doped mesoporous bioactive glass (Mg-MBG) according to the proportion of 200g:20Mg for 1h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 24h, centrifuging at 12,000rpm for 5min, and taking the precipitate to obtain Mg-MBG material (OOB @ Mg-MBG) loaded with the OOB fusion protein;
2) passing the silkworm cocoon through Na according to the ratio of 6g to 2.2g/L 2 CO 3 Boiling the aqueous solution for degumming, then placing the aqueous solution in a 50 ℃ oven for drying, and completely dissolving the aqueous solution in 8M lithium bromide in the 50 ℃ oven to obtain dissolved silk fibroin;
dialyzing the dissolved silk fibroin by deionized water, and concentrating by 10% PEG solution with molecular weight of 10000 for 60 hours;
finally centrifuging the concentrated silk fibroin for 2 times at 4 ℃ and 8000rpm for 20min each time, and removing impurities to finally obtain a silk fibroin original solution;
3) mixing and stirring neurotrophic factor (NTF) and Silk Fibroin (SF) solution according to the proportion of 6 mug to 20mg for 1h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 24h, centrifuging the mixture at 12,000rpm for 5min, and taking a precipitate to obtain an NTF-loaded SF material (NTF @ SF);
4) placing poly-epsilon-caprolactone (PCL) in hexafluoroisopropanol solution, and magnetically stirring until the solution is completely clear according to the proportion of 5g to 50mL to obtain a spinning solution;
and (2) adding the OOB @ Mg-MBG obtained in the step 1) and the NTF @ SF obtained in the step 3) into a spinning solution according to the concentration of 5-15Mg/mL respectively, taking the OOB @ Mg-MBG as an inner core and the NTF @ SF as an outer shell, stirring and ultrasonically mixing the solutions sufficiently, and performing electrostatic spinning in a high-voltage direct-current electric field of 22kV at the speed of 0.70mL/h, the temperature of 25 ℃ and the relative humidity of 44% by using a digital injection pump to obtain the bone repair scaffold material capable of mediating the neurogenesis.
Example 2:
a preparation method of a bone repair scaffold material capable of mediating the neurogenesis comprises the following steps:
1) mixing and stirring OCN-OPN-BGN fusion protein (OOB fusion protein) heterologously expressed in vitro and magnesium-doped mesoporous bioactive glass (Mg-MBG) according to the proportion of 100g:20Mg for 2h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 48h, centrifuging at 12,000rpm for 5min, and taking the precipitate to obtain Mg-MBG material (OOB @ Mg-MBG) loaded with the OOB fusion protein;
2) passing the silkworm cocoon through Na according to the proportion of 3g to 4.4g/L 2 CO 3 Boiling the aqueous solution for degumming, then placing the aqueous solution in a drying oven at 40 ℃ for drying, and completely dissolving the aqueous solution in a 10M lithium bromide solution in the drying oven at 40 ℃ to obtain dissolved silk fibroin;
dialyzing the dissolved silk fibroin by deionized water, and concentrating for 48 hours by using 5% PEG solution with the molecular weight of 10000;
finally centrifuging the concentrated silk fibroin for 2 times at 4 ℃ and 7000rpm for 20min each time, and removing impurities to finally obtain a silk fibroin original solution;
3) mixing and stirring neurotrophic factor (NTF) and Silk Fibroin (SF) solution according to the proportion of 5 mug to 10mg for 2h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 48h, centrifuging the mixture at 12,000rpm for 5min, and taking a precipitate to obtain an NTF-loaded SF material (NTF @ SF);
4) placing poly-epsilon-caprolactone (PCL) in a hexafluoroisopropanol solution, and magnetically stirring until the solution is completely clear according to the proportion of 4g to 50mL to obtain a spinning solution;
and (2) adding the OOB @ Mg-MBG obtained in the step 1) and the NTF @ SF obtained in the step 3) into a spinning solution according to the concentration of 5-15Mg/mL respectively, taking the OOB @ Mg-MBG as an inner core and the NTF @ SF as an outer shell, stirring and ultrasonically mixing the solutions sufficiently, and performing electrostatic spinning under a high-voltage direct-current electric field of 18kV and a digital injection pump at the speed of 0.60mL/h, the temperature of 20 ℃ and the relative humidity of 60% to obtain the bone repair scaffold material capable of mediating the neurogenesis.
Example 3:
a preparation method of a bone repair scaffold material capable of mediating the neurogenesis comprises the following steps:
1) mixing and stirring OCN-OPN-BGN fusion protein (OOB fusion protein) heterologously expressed in vitro and magnesium-doped mesoporous bioactive glass (Mg-MBG) according to the proportion of 300g to 20Mg for 1.5h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 32h, centrifuging at 12,000rpm for 5min, and taking precipitate to obtain Mg-MBG material (OOB @ Mg-MBG) loaded with the OOB fusion protein;
2) passing the silkworm cocoon through Na according to the ratio of 8g to 3.3g/L 2 CO 3 Boiling the aqueous solution for degumming, then placing the aqueous solution in an oven at 80 ℃ for drying, and completely dissolving the aqueous solution in a 7M lithium bromide solution in the oven at 80 ℃ to obtain dissolved silk fibroin;
dialyzing the dissolved silk fibroin by deionized water, and concentrating for 12 hours by 20% PEG solution with the molecular weight of 10000;
finally centrifuging the concentrated silk fibroin for 2 times at 12000rpm at 4 ℃ for 20min each time, and removing impurities to finally obtain a silk fibroin original solution;
3) mixing and stirring neurotrophic factor (NTF) and Silk Fibroin (SF) solution according to the proportion of 8 mug to 30mg for 1.5h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 32h, centrifuging the mixture at 12,000rpm for 5min, and taking the precipitate to obtain an NTF-loaded SF material (NTF @ SF);
4) placing poly-epsilon-caprolactone (PCL) in hexafluoroisopropanol solution, and magnetically stirring until the solution is completely clear according to the proportion of 6g to 50mL to obtain a spinning solution;
adding the OOB @ Mg-MBG obtained in the step 1) and the NTF @ SF obtained in the step 3) into a spinning solution according to the concentration of 5-15Mg/mL respectively, taking the OOB @ Mg-MBG as an inner core and the NTF @ SF as an outer shell, fully and uniformly mixing the solution through stirring and ultrasound, and performing electrostatic spinning in a high-voltage direct-current electric field of 20kV by using a digital injection pump at the speed of 0.85mL/h, the temperature of 24 ℃ and the relative humidity of 30% to obtain the bone repair scaffold material capable of mediating the neurogenesis.
Experimental example:
characterization measurements and results of one bone repair scaffold material that can mediate neurogenesis obtained in example 1:
fig. 1 is a microscopic surface structure of a bone repair scaffold material capable of mediating the neural transformation obtained in example 1 by scanning electron microscopy.
And spraying gold for 30s on the Mg-MBG, the OOB @ Mg-MBG and the bone repair scaffold, opening the system, putting a sample to be detected into a sample chamber, closing the sample chamber, vacuumizing and observing, wherein the Mg-MBG, the OOB @ Mg-MBG and the bone repair scaffold capable of mediating the neurogenesis all have uniform scaffold structures as shown in figure 1.
FIG. 2 is the Mg-MBG, OOB @ Mg-MBG and fluid simulating human body fluid (SBF) (142.0mM Na) of the bone repair scaffold capable of mediating neurogenesis of example 1 + ，5.0mM K + ，1.5mM Mg 2+ ，2.5mM Ca 2+ ，103.0mM Cl - ，27.0mM HCO 3- ，1.0mM HPO 4 2- ，0.5mM SO 4 2- ) The degree of mineralization in vitro was tested using a scanning electron microscope image.
The reaction temperature of the experiment is 37 ℃, and after mineralization, a scanning electron microscope is adopted for observation and shows that OOB @ Mg-MBG and the bone repair scaffold capable of mediating the neurogenesis are mineralized in vitro more than the Mg-MBG scaffold, and the form of the mineralized deposit is similar to that of calcium deposit, so that the bone repair scaffold capable of mediating the neurogenesis can be subjected to a good mineralization process in vitro.
FIG. 3 is a graph showing the measurement of the proliferation potency of mesenchymal stem cells of Mg-MBG, OOB @ Mg-MBG and bone repair scaffold material capable of mediating neurogenesis in example 1, measured by Cell Counting Kit-8 (CCK-8), and the absorbance value at a wavelength of 450nm using a microplate reader.
According to the instruction, CCK-8 is added according to 10% of the volume of the culture solution 4 hours before the test, and the proliferation of the cells is measured by reading the absorbance value at the wavelength of 450nm of an enzyme labeling instrument when the cells grow for 1, 3, 5 and 7 days, and the result shows that the bone repair scaffold material capable of mediating the neurogenesis promotes the proliferation of MC3T3-E1 cells.
FIG. 4 is a graph in which the differentiation ability of Mg-MBG, OOB @ Mg-MBG and the bone repair scaffold material mediating the neurogenesis in example 1 to mesenchymal stem cells is measured by measuring the activity of alkaline phosphatase, a prophase index of cell differentiation.
In the assay, cells were assayed in two separate groups, one group using p-nitrophenol phosphate (pNPP) working solution (8 mM pNPP, 0.1% Triton X-100, 2mM MgCl) 2 ，0.1M Na 2 CO 3 -NaHCO 3 Buffer (pH 10.3)), added to cells in an amount of 100. mu.l per well, incubated at 37 ℃ for 30 minutes, and then measured for absorbance at 405nm by a microplate reader, and another set was used for measuring the intracellular protein concentration by the bicinchoninic acid (BCA) -based method as an internal reference, added to 200. mu.l per well as described in the specification, incubated at 37 ℃ for 30 minutes, and then measured for absorbance at 550nm by a microplate reader, and alkaline phosphatase activity = A was calculated according to the formula 405 /A 550 。
The results show (FIG. 4) that bone marrow mesenchymal stem cell culture was highest in alkaline phosphatase activity, next to OOB @ Mg-MBG scaffold, lowest in Mg-MBG scaffold in bone repair scaffold that could mediate neurogenesis; therefore, the differentiation capability of the bone repair scaffold material capable of mediating the neurogenesis on osteoblasts is strongest, the OOB @ Mg-MBG scaffold is second, and the Mg-MBG scaffold is lowest.
FIG. 5 is a graph of the mineralization capacity of Mg-MBG, OOB @ Mg-MBG and bone repair scaffold material mediating neurogenesis in example 1 on bone marrow mesenchymal stem cells as determined by alizarin red-stained mineralized nodules.
The mesenchymal stem cells of the bone marrow are 5 multiplied by 10 5 one/mL of the culture medium was cultured in the scaffold material continuously for 3 days, and then the culture medium was changed to a mineralized medium (α MEM culture medium containing 10mM β -sodium glycerophosphate, 50 μ g/mL vitamin C, 10% FBS); and (3) carrying out determination on mineralized nodules on the 14 th day of cell culture, specifically fixing the cells at room temperature for 10min by using 75% ethanol, dyeing the mineralized nodules for 10min by using alizarin red working solution (pH 4.2 and 40 mM) at 37 ℃, cleaning redundant working solution by using deionized water, drying and taking pictures. The larger the number of nodules, the better the mineralization.
The results are shown in fig. 5, the mineralization capacity of bone repair scaffold material capable of mediating the neurogenesis is maximum on bone marrow mesenchymal stem cells, OOB @ Mg-MBG scaffold is second, and Mg-MBG scaffold is minimum.
FIG. 6 is a graph of the ability of Mg-MBG, OOB @ Mg-MBG, and bone repair scaffold materials that mediate neurogenesis in example 1 to differentiate bone marrow mesenchymal stem cells into the neural direction as measured by immunofluorescence.
Bone marrow mesenchymal stem cells are respectively induced on three scaffold materials, fixed for 30 minutes at room temperature by adopting methanol-acetone (1: 1), blocked overnight at 4 ℃ through 10% horse serum, diluted by anti-NSE and anti-GFAP antibodies in a ratio of 1:100, incubated overnight at 4 ℃, then diluted by FITC and Alexa Fluor 594 labeled secondary antibodies in a ratio of 1:100, incubated for 2 hours at room temperature, and after staining cell nuclei for 5 minutes, the types, the quantity and the forms of nerve cells are observed under a microscope (Leica, Germany) with ultraviolet, blue light and excitation green light waves.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modifications, equivalent substitutions, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention are within the scope of the technical scheme of the present invention.
Claims (3)
1. A preparation method of a bone repair scaffold material capable of mediating the neurogenesis is, which is characterized by comprising the following steps: the method comprises the following steps:
1) mixing and stirring OCN-OPN-BGN fusion protein heterologously expressed in vitro and the magnesium-doped mesoporous bioactive glass according to the proportion of (100) g:20Mg for 1-2h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorption for 24-48h, centrifuging at 12,000rpm for 5min, and taking the precipitate to obtain Mg-MBG material loaded with OOB fusion protein;
2) according to the proportion of (3-8) g (2.2-4.4) g/L, enabling the silkworm cocoons to pass through Na 2 CO 3 Boiling the water solution for degumming, drying in an oven at 40-80 deg.C, and completely dissolving in an oven at 40-80 deg.C with 7-10M lithium bromide solution to obtain dissolved silk fibroin;
dialyzing the dissolved silk fibroin by deionized water, and concentrating by 5-20% PEG solution with molecular weight of 10000 for 12-60 hours;
finally centrifuging the concentrated silk fibroin for 2 times at 4 ℃, 7000-12000rpm for 20min each time, removing impurities, and finally obtaining a silk fibroin original solution;
3) mixing and stirring neurotrophic factors and silk fibroin solution according to the proportion of (5-8) mu g (10-30) mg for 1-2h, placing the mixture in a refrigerator at 4 ℃ for standing and adsorbing for 24-48h, centrifuging at 12,000rpm for 5min, and taking the precipitate to obtain the SF material loaded with NTF;
4) placing poly-epsilon-caprolactone in hexafluoroisopropanol solution, magnetically stirring according to the proportion of (4-6) g:50mL until the solution is completely clear, and performing electrostatic spinning by using a digital injection pump at the speed of 0.70mL/h, the temperature of 25 ℃ and the relative humidity of 44% to obtain a spinning solution;
adding the Mg-MBG material loaded with the OOB fusion protein obtained in the step 1) and the SF material loaded with the NTF obtained in the step 3) into a spinning solution according to 5-15Mg/mL respectively, taking the Mg-MBG material loaded with the OOB fusion protein as an inner core and the SF material loaded with the NTF as an outer shell, fully and uniformly mixing the solutions by stirring and ultrasonic treatment, and performing electrostatic spinning under a high-voltage direct-current electric field of 18-22kV and a digital injection pump at the speed of 0.60-0.85mL/h, the temperature of 20-25 ℃ and the relative humidity of 30-60% to obtain the bone repair support material capable of mediating the neurogization.
2. A bone repair scaffold material capable of mediating neurogenesis, comprising: the preparation method of the bone repair scaffold material capable of mediating the neurogenesis, which is disclosed in claim 1.
3. Use of a bone repair scaffold material according to claim 2 for mediating neurogenesis in the preparation of a product for the repair of a bone defect.
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