CN110180029B - Preparation method and application of degradable material with osteogenic differentiation and bone regeneration induction functions - Google Patents
Preparation method and application of degradable material with osteogenic differentiation and bone regeneration induction functions Download PDFInfo
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Abstract
The invention discloses a micro RNA-214 inhibitor loaded degradable material with functions of inducing osteogenic differentiation and bone regeneration, which is prepared from the following raw materials: graphene oxide, polyethyleneimine, micro RNA-214 inhibitor, silk fibroin and nano hydroxyapatite. The micro RNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration is degradable in vivo, has a good osteogenic differentiation function and higher bioactivity, has obvious advantages in the aspects of promoting cell proliferation and differentiation, promoting cell adhesion and the like, and has no obvious influence on the surrounding environment by degradation products. Therefore, the load microRNA-214 inhibitor degradable material with the functions of inducing osteogenic differentiation and bone regeneration is suitable for minimally invasive bone repair application in clinic.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a preparation method and application of a degradable material with functions of inducing osteogenic differentiation and bone regeneration.
Background
Currently, bone defects have become the most common disease in the clinic. For patients with large bone defect area, the self-healing effect is often difficult to achieve depending on the regeneration and repair capability of bones due to the limitation of factors such as destruction or repair environment. The substitute material is a common method for repairing large-area bone defects and has wide application prospect. Bone tissue engineering is a promising approach which provides a suitable technique for designing bone substitutes/regenerating new bone tissue for fracture repair due to traffic accidents, traumatic injuries, bone diseases, tumor resection, bone necrosis or rheumatism. However, bone substitutes/grafts (allo/autologous) are limited by the lack of ideal bone substitutes, osteogenic potential of the graft, immune rejection and secondary trauma. The micro RNA-214 can target a transcription factor ATF4 in osteoblasts to inhibit bone formation, and the micro RNA-214 inhibitor has the effects of promoting osteogenesis, inhibiting the bidirectional regulation of bone activity and activating AKT and ERK1/2 signal pathways of the osteoblasts so as to promote the differentiation of the cells. Aiming at the problems, a degradable three-dimensional scaffold loaded with a micro RNA-214 inhibitor and having the functions of inducing osteogenic differentiation and bone regeneration is researched. The invention analyzes the degradable material with the bone repair function from two aspects of osteogenic differentiation and bone regeneration. The result shows that the degradable three-dimensional scaffold loaded with the micro RNA-214 inhibitor and having the functions of inducing osteogenic differentiation and bone regeneration can promote cell adhesion and effectively promote osteogenic differentiation and bone regeneration.
The clinical treatment means for bone defects mainly comprises bone grafting and bone substitute materials. Wherein the bone graft includes autologous bone, allogeneic bone and xenogeneic bone graft. Autologous bone transplantation, which does not produce immune rejection and has good osteoinductive and osteoconductive properties, has extremely limited sources; although the source of the allogeneic bone and the xenogeneic bone is rich, the xenogeneic bone and the xenogeneic bone can generate immunological rejection reaction and also have complications such as infection and the like and have poor osteoinduction effect.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and prepare a micro RNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration, which can promote cell adhesion, osteogenic differentiation and bone formation, is superior to the traditional bone graft and the traditional scaffold, and has the degradation speed matched with the bone repair speed.
In order to achieve the purpose, the invention adopts the technical scheme that:
a micro RNA-214 inhibitor loaded degradable material with functions of inducing osteogenic differentiation and bone regeneration is characterized by comprising the following raw materials: graphene oxide, polyethyleneimine, micro RNA-214 inhibitor, silk fibroin and nano hydroxyapatite.
The invention also provides a preparation method of the micro RNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration, which comprises the following steps:
(1) preparing graphene oxide and polyethyleneimine into a solution with a certain concentration, mixing, performing ultrasonic treatment, and stirring to obtain a graphene oxide-polyethyleneimine compound solution;
(2) compounding the compound solution obtained in the step (1) with a micro RNA-214 inhibitor in a phosphate buffer solution, vortexing at room temperature, and incubating to obtain a graphene oxide-polyethyleneimine compound solution loaded with the micro RNA-214 inhibitor;
(3) mixing and stirring an ammonium hydrogen phosphate solution and a calcium chloride solution, aging and centrifuging at room temperature, and freeze-drying the obtained precipitate to obtain nano-hydroxyapatite particles; drying the silk fibroin extracted from the silkworm cocoon, placing the dried silk fibroin in a lithium bromide solution, heating and dissolving to obtain a silk fibroin solution, dialyzing, and centrifugally collecting for later use;
(4) and (3) mixing and stirring the silk fibroin solution with the concentration of the step (3), nano hydroxyapatite and the graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor compound in the step (2), adding the mixture into a pore plate, and freeze-drying to obtain the degradable material with the functions of inducing osteogenic differentiation and bone regeneration.
Preferably, the concentration of the graphene oxide in the step (1) is 0.1-10 mg/mL; the concentration of the polyethyleneimine is 0.1-10 mg/mL.
Preferably, the concentration of the graphene oxide in the step (1) is 1 mg/mL; the concentration of polyethyleneimine was 1 mg/mL.
Preferably, the mass ratio of the graphene oxide to the polyethyleneimine in the step (1) in the reaction system is 1: 1-5.
Preferably, the mass ratio of the graphene oxide to the polyethyleneimine in the step (1) in the reaction system is 1: 2.
Preferably, the ultrasonic time in the step (1) is 10-60 min; the stirring time is 3-24 h.
Preferably, the ultrasound time in the step (1) is 15 min; the stirring time was 12 h.
Preferably, the mass ratio of the graphene oxide-polyethyleneimine complex to the micro RNA-214 inhibitor in the step (2) is 100-30: 1.
Preferably, the mass ratio of the graphene oxide-polyethyleneimine complex to the micro RNA-214 inhibitor in the step (2) is 30: 1.
Preferably, the vortex time in the step (2) is 30-100 s; the incubation time is 10-120 min.
Preferably, the vortex time in the step (2) is 60-100 s; the incubation time is 60 min.
Preferably, the concentration of the ammonium hydrogen phosphate in the reaction system in the step (3) is 0.1-1 mol/L; the concentration of calcium chloride in the reaction system is 0.1-1 mol/L; the concentration of lithium bromide in the reaction system is 1-20 mol/L; the mass fraction of the silk fibroin in the reaction system is 1-10%.
Preferably, the concentration of the ammonium hydrogen phosphate in the reaction system in the step (3) is 0.3 mol/L; the concentration of calcium chloride in the reaction system is 0.5 mol/L; the concentration of lithium bromide in the reaction system is 9.3 mol/L; the mass fraction of the silk fibroin in the reaction system is 4%.
Preferably, the stirring time in the step (3) is 1-6 h; aging and centrifuging for 12-36 h; the centrifugal speed is 1000-10000 rpm, and the centrifugal time is 5-60 min; the heating time is 1-12 h, and the heating temperature is 30-100 ℃.
Preferably, the stirring time in the step (3) is 2 h; aging and centrifuging for 24 hours; the centrifugal speed is 5000rpm, and the centrifugal time is 10 min; the heating time is 5h, and the heating temperature is 60 ℃.
Preferably, the concentration of the graphene oxide-polyethyleneimine-micro RNA-214 inhibitor compound in the step (4) in a system is 0.1-10 mg/mL.
Preferably, the concentration of the graphene oxide-polyethyleneimine-micro RNA-214 inhibitor complex in the step (4) in the system is 1 mg/mL.
The invention also aims to provide application of the micro RNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration in bone repair.
The invention has the beneficial effects that: the invention provides a micro RNA-214 inhibitor loaded degradable material with functions of inducing osteogenic differentiation and bone regeneration, which consists of a bracket consisting of nano hydroxyapatite and silk fibroin and a graphene oxide loaded micro RNA-214 inhibitor compound modified by polyethyleneimine. The micro RNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration is degradable in vivo, has a good osteogenic differentiation function and higher bioactivity, has obvious advantages in the aspects of promoting cell proliferation and differentiation, promoting cell adhesion and the like, and has no obvious influence on the surrounding environment by degradation products. Therefore, the load microRNA-214 inhibitor degradable material with the functions of inducing osteogenic differentiation and bone regeneration is suitable for minimally invasive bone repair application in clinic.
Drawings
Fig. 1 is an infrared spectrum of graphene oxide, polyethyleneimine and polyethyleneimine-modified graphene oxide.
Fig. 2 is a transmission electron microscope image of graphene oxide and polyethyleneimine-modified graphene oxide.
Fig. 3 is a schematic diagram illustrating the results of gel retardation experiments on the degradable material having the functions of inducing osteogenic differentiation and bone regeneration according to the present invention.
Fig. 4 is a schematic diagram of the cell activity of the degradable material with osteogenic differentiation and bone regeneration inducing function according to the present invention.
FIG. 5 is a schematic diagram showing the activity of the degradable material alkaline phosphatase (ALP) having the function of inducing osteogenic differentiation and bone regeneration according to the present invention.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.
the method comprises the following steps: synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene Oxide (GO) is prepared into 1mg/mL for standby after ultrasonic treatment, and polyethyleneimine (25k) (PEI) is prepared into a 1mg/mL solution and is slowly added into the GO solution within 10 min. The GO-PEI complex solution is obtained by mixing a GO solution and a diluted PEI solution according to the mass ratio of 1:2, then carrying out ultrasonic treatment for 15min, then stirring for 12 hours overnight, and then removing free PEI through centrifugation and washing.
Step two: synthesis of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor complex (GO-PEI-microRNA-214)
Compounding the GO-PEI compound solution and the micro RNA-214 inhibitor in a phosphate buffer solution according to the mass ratio of 30:1, vortexing for 60s at room temperature, and incubating for 60min to obtain the graphene oxide-polyethyleneimine compound loaded with the micro RNA-214 inhibitor. Wherein: specific information of the micro RNA-214 inhibitor is as follows: name of miRNA: mmu-miR-214-3p miRBase Access: MIMAT0000661 miRNA mature sequence: acagcaggcacagacaggcagu are provided.
Step three: preparation of silk fibroin and nano hydroxyapatite
Mixing and stirring an ammonium hydrogen phosphate solution with the concentration of 0.3mol/L and a calcium chloride solution with the concentration of 0.5mol/L, aging and centrifuging for 24 hours at room temperature (pH is 10), and freeze-drying the obtained precipitate for 24 hours to obtain the nano-hydroxyapatite particles. And drying the silk fibroin extracted from the silkworm cocoon, placing the dried silk fibroin in a lithium bromide solution with the concentration of 9.3mol/mL, heating and dissolving for 5 hours at the temperature of 60 ℃ to obtain a silk fibroin solution, dialyzing for 3d, and centrifuging at 5000rpm for 10min for collection for later use.
Step four: synthesis of degradable material with osteogenic differentiation and bone regeneration inducing function
Mixing and stirring 1mL of silk fibroin solution with the concentration of 4% (w/v), 10mg of nano-hydroxyapatite and 1mL of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor composite solution with the concentration of 1mg/mL, adding the mixture into a 24-pore plate, and freeze-drying to obtain the degradable material with the functions of inducing osteogenic differentiation and bone regeneration.
Embodiment 2 the preparation method of the degradable material loaded with the micro RNA-214 inhibitor comprises the following steps:
the method comprises the following steps: synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene Oxide (GO) was formulated to 0.1mg/mL for use after sonication, and polyethyleneimine (25k) (PEI) was formulated as a 0.1mg/mL solution and slowly added to the GO solution over 10 min. The GO-PEI complex solution is obtained by mixing a GO solution and a diluted PEI solution according to the mass ratio of 1:1, then carrying out ultrasonic treatment for 15min, then stirring for 12 hours overnight, and then removing free PEI through a centrifugation and washing method.
Step two: synthesis of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor complex (GO-PEI-microRNA-214)
Compounding the GO-PEI compound solution and the micro RNA-214 inhibitor in a phosphate buffer solution according to the mass ratio of 100:1, vortexing for 60s at room temperature, and incubating for 60min to obtain the graphene oxide-polyethyleneimine compound loaded with the micro RNA-214 inhibitor.
Step three: preparation of silk fibroin and nano hydroxyapatite
Mixing and stirring an ammonium hydrogen phosphate solution with the concentration of 0.1mol/L and a calcium chloride solution with the concentration of 0.1mol/L, aging and centrifuging for 24 hours at room temperature (pH is 10), and freeze-drying the obtained precipitate for 24 hours to obtain the nano-hydroxyapatite particles. And drying the silk fibroin extracted from the silkworm cocoon, placing the dried silk fibroin in a lithium bromide solution with the concentration of 1mol/mL, heating and dissolving for 5 hours at the temperature of 60 ℃ to obtain a silk fibroin solution, dialyzing for 3d, and centrifuging at 5000rpm for 10min for collection for later use.
Step four: synthesis of degradable material with osteogenic differentiation and bone regeneration inducing function
Mixing and stirring 1mL of silk fibroin solution with the concentration of 1% (w/v), 10mg of nano-hydroxyapatite and 1mL of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor composite solution with the concentration of 0.1mg/mL, adding the mixture into a 24-pore plate, and freeze-drying to obtain the degradable material with the functions of inducing osteogenic differentiation and bone regeneration.
the method comprises the following steps: synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene Oxide (GO) is prepared into 6mg/mL for standby after ultrasonic treatment, and polyethyleneimine (25k) (PEI) is prepared into 6mg/mL solution and is slowly added into the GO solution within 10 min. The GO-PEI complex solution is obtained by mixing a GO solution and a diluted PEI solution according to the mass ratio of 1:4, then carrying out ultrasonic treatment for 15min, then stirring for 12 hours overnight, and then removing free PEI through centrifugation and washing.
Step two: synthesis of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor complex (GO-PEI-microRNA-214)
Compounding the GO-PEI compound solution and the micro RNA-214 inhibitor in a phosphate buffer solution according to the mass ratio of 60:1, vortexing for 60s at room temperature, and incubating for 60min to obtain the graphene oxide-polyethyleneimine compound loaded with the micro RNA-214 inhibitor.
Step three: preparation of silk fibroin and nano hydroxyapatite
Mixing and stirring an ammonium hydrogen phosphate solution with the concentration of 0.6mol/L and a calcium chloride solution with the concentration of 0.8mol/L, aging and centrifuging for 24 hours at room temperature (pH is 10), and freeze-drying the obtained precipitate for 24 hours to obtain the nano-hydroxyapatite particles. And drying the silk fibroin extracted from the silkworm cocoon, placing the dried silk fibroin in a lithium bromide solution with the concentration of 5mol/mL, heating and dissolving for 5 hours at the temperature of 60 ℃ to obtain a silk fibroin solution, dialyzing for 3d, and centrifuging at 5000rpm for 10min for collection for later use.
Step four: synthesis of degradable material with osteogenic differentiation and bone regeneration inducing function
Mixing and stirring 1mL of silk fibroin solution with the concentration of 8% (w/v), 10mg of nano-hydroxyapatite and 1mL of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor composite solution with the concentration of 5mg/mL, adding the mixture into a 24-pore plate, and freeze-drying to obtain the degradable material with the functions of inducing osteogenic differentiation and bone regeneration.
Embodiment 4 the preparation method of the degradable material loaded with the micro RNA-214 inhibitor comprises the following steps:
the method comprises the following steps: synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene Oxide (GO) is prepared into 10mg/mL for standby after ultrasonic treatment, and polyethyleneimine (25k) (PEI) is prepared into 10mg/mL solution and is slowly added into the GO solution within 10 min. The GO-PEI complex solution is obtained by mixing a GO solution and a diluted PEI solution according to the mass ratio of 1:5, then carrying out ultrasonic treatment for 15min, then stirring for 12 hours overnight, and then removing free PEI through a centrifugation and washing method.
Step two: synthesis of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor complex (GO-PEI-microRNA-214)
Compounding the GO-PEI compound solution and the micro RNA-214 inhibitor in a phosphate buffer solution according to the mass ratio of 90:1, vortexing for 60s at room temperature, and incubating for 60min to obtain the graphene oxide-polyethyleneimine compound loaded with the micro RNA-214 inhibitor.
Step three: preparation of silk fibroin and nano hydroxyapatite
Mixing and stirring a 1mol/L ammonium hydrogen phosphate solution and a 1mol/L calcium chloride solution, aging and centrifuging for 24 hours at room temperature (pH 10), and freeze-drying the obtained precipitate for 24 hours to obtain nano-hydroxyapatite particles. And drying the silk fibroin extracted from the silkworm cocoon, placing the dried silk fibroin in a lithium bromide solution with the concentration of 20mol/mL, heating and dissolving for 5 hours at the temperature of 60 ℃ to obtain a silk fibroin solution, dialyzing for 3d, and centrifuging at 5000rpm for 10min for collection for later use.
Step four: synthesis of degradable material with osteogenic differentiation and bone regeneration inducing function
Mixing and stirring 1mL of silk fibroin solution with the concentration of 10% (w/v), 10mg of nano-hydroxyapatite and 1mL of graphene oxide-polyethyleneimine loaded micro-RNA-214 inhibitor composite solution with the concentration of 10mg/mL, adding the mixture into a 24-pore plate, and freeze-drying to obtain the degradable material with the functions of inducing osteogenic differentiation and bone regeneration.
Example 5: synthesis and infrared analysis of graphene oxide-polyethyleneimine (GO-PEI)
The FTIR Fourier infrared spectrum is a spectrum for displaying molecular vibration, can identify functional groups in a substance to be detected, and represents the characteristic peak changes of GO, PEI and GO-PEI through the Fourier infrared spectrum. And infrared tests are carried out on the GO-PEI, the GO and the PEI to prove whether the target product is successfully synthesized. Respectively taking 1-2mg of GO and PEI and the GO-PEI samples of example 1, placing the samples in an agate mortar for grinding, sequentially adding 100-200mg of potassium bromide powder after drying for 24 hours (the proportion of the sample to be tested in the potassium bromide is about 0.5% -1%), continuously grinding until the granularity is fine, mixing, and obtaining the productAfter the mixture is uniformly mixed, the mixture is pressed into a transparent sheet for testing under the vacuum degree of 10mmHg for 2-5min, and the scanning range is 4000--1The results obtained are shown in FIG. 1. Fourier transform infrared (FT-IR) analysis shows that GO and GO-PEI both have-OH, C ═ O and C-O groups, corresponding to 3200--1、1500-1760cm-1And 1000--1Peak value of (a). C-H, N-H and C-N vibration peaks of PEI and GO-PEI respectively correspond to 500-900cm-1、1000-1250cm-1And 1250--1. The results of these peaks indicate that PEI is grafted to graphene oxide via electrostatic interactions rather than covalent bonds, demonstrating the successful synthesis of the GO-PEI complex in example 1.
Example 6: synthesis of graphene oxide-polyethyleneimine (GO-PEI) and analysis of transmission electron microscope
The microscopic morphology of the GO-PEI composite particles prepared in the first step of example 1 was observed by Transmission Electron Microscopy (TEM). Analysis of a transmission electron microscope in fig. 2 shows that the surface of graphene oxide is relatively smooth, and the PEI functionalized graphene oxide changes the smoothness of the surface of the graphene oxide and increases the size of the graphene oxide.
Example 7: gel blocking of GO-PEI and micro RNA-214 inhibitor
And (3) rapidly adding the micro RNA-214 inhibitor into the graphene oxide-polyethyleneimine mother liquor obtained in the embodiment 1, slowly reversing the micro RNA-214 inhibitor from top to bottom for 8-10 times, and incubating for 30min at 25 ℃ for later use. A series of nanocomposite complexes of different mass ratios (GO-PEI/micro RNA-214) were prepared, setting the mass ratios to 0, 10, 20, 30, 40, 50 and 60. By changing the adding amount of each group of GO-PEI, the adding amount of each group of micro RNA-214 inhibitor is kept consistent so as to be used for the detection of the subsequent gel retardation experiment. Then 1% agarose gel containing 0.1% EB dye solution is prepared, each group of prepared samples are added into the pore channel gently, and the gel is immersed by TAE buffer solution. The voltage was set at 100V, electrophoresis was started, and after 25min, electrophoresis was stopped. The agarose gel was carefully removed, and after UV irradiation, it was photographed using a gel imaging system, and the results are shown in FIG. 3. Experimental results show that when the mass ratio of the GO-PEI to the micro RNA-214 inhibitor is less than or equal to 20, the micro RNA-214 inhibitor and the GO-PEI are not completely combined, and when the mass ratio of the GO-PEI to the micro RNA-214 inhibitor reaches 30, the micro RNA-214 inhibitor and the GO-PEI are blocked at a sample loading hole, and the micro RNA-214 inhibitor and the GO-PEI form a compact nano composite structure. Therefore, GO-PEI/micro RNA-214 with the mass ratio of 30 is selected in subsequent experiments.
Example 8: cell activity detection made of degradable material loaded with microRNA-214 inhibitor and having functions of inducing osteogenic differentiation and bone regeneration
The cell activity of the degradable material (SF/HAP/GPM) prepared in example 1 was examined by CCK-8 method, and silk fibroin Scaffold (SF) and silk fibroin-nano hydroxyapatite composite scaffold (SF/HAP) were used as control. The cells used in this experiment were fibroblasts (3T3 cells), and the culture medium used for culturing the cells was DMEM containing 10% fetal bovine serum and 1% diabody (mixture of penicillin and streptomycin), and the culture conditions were at 37 ℃ and CO 25% concentration incubator. During the culture process, the cells are changed every two days with the aim of adding new nutrients to the cells, removing nonadherent cells and metabolites of the cells. And placing the sterilized materials of different groups into a 48-well plate, then dripping 50 mu L of adjusted cell suspension onto the microRNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration, incubating for 2h in an incubator, adding 450 mu L of culture solution onto the material, and continuing to culture. After 3 and 7 days of culture, CCK-8 reagent is added according to the proportion of 1:10, namely 10 mu L of CCK-8 reagent is added into 100 mu L of culture solution, and the culture is continued for 2-4 h. The absorbance of each well was read using a microplate reader at a wavelength of 450 nm. The results are shown in FIG. 4 and show that: the cell proliferation rate of the SF/HAP/GPM scaffold is higher than that of the SF and the SF/HAP scaffold. On day 3, cell proliferation reached a higher level; however, at day 7, proliferation of cells decreased, and cells tended to differentiate and mineralize. The results show that the degradable material loaded with the microRNA-214 inhibitor and having the functions of inducing osteogenic differentiation and bone regeneration has good biocompatibility and cell adhesion.
Example 9: alkaline phosphatase (ALP) activity detection of load microRNA-214 inhibitor degradable material with osteogenic differentiation and bone regeneration induction functions
Respectively placing sterilized load microRNA-214 inhibitor degradable material (SF/HAP/GPM) with osteogenic differentiation and bone regeneration induction functions, silk fibroin bracket material (SF) and silk fibroin-hydroxyapatite bracket material (SF/HAP) in a 48-hole culture plate. The cells cultured for 3 passages were removed from the flask by digestion with 0.25% pancreatin, centrifuged at 1000rpm for 5min, the supernatant was discarded, and an α -DMEM medium containing serum and diabody (a mixture of penicillin and chain) was added thereto at a cell concentration of 5X 10 cells/ml7And (4) cells. Each sample was seeded with 20. mu.L of the above cell suspension and placed at 37 ℃ in 5% CO2The incubator of (4) was incubated in the incubator for 2 hours, and further 500. mu.L of the culture solution was added thereto to continue the culture for 7 days, 14 days and 21 days. During the culture period, the culture medium was changed once for 2 to 3 days in order for the cells to obtain sufficient nutrients. Taking out the material from the pore plate, rinsing the microRNA-214 inhibitor-loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration for 3 times by using sterile PBS solution, adding 500 mu L of cell lysate into the material, and then placing the material in an ultrasonic cell disruption instrument at the temperature of 4 ℃ for cell disruption. This was centrifuged and the supernatant was collected. To the supernatant was added 500. mu.L of the ALP substrate reaction solution, and reacted at 37 ℃ in a water bath for 30min, to terminate the reaction, 500. mu.L of NaOH having a concentration of 0.1M was added to the reaction solution, followed by measuring the spectrophotometric value of the sample at 405nm with an ultraviolet-visible spectrophotometer, and ALP was calculated with the aid of the specification. Each set of materials at each time point was tested at least 3 times in parallel. The experimental result is shown in fig. 5, the ALP activity of the cells in the microRNA-214 inhibitor-loaded degradable material with the function of inducing osteogenic differentiation and bone regeneration tends to increase along with the prolongation of the incubation time within 14 days, and the result shows that the microRNA-214 inhibitor-loaded degradable material with the function of inducing osteogenic differentiation and bone regeneration is beneficial to osteogenic differentiation and is superior to the material without the microRNA-214 inhibitorOsteogenic differentiation ability of.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. A micro RNA-214 inhibitor loaded degradable material with functions of inducing osteogenic differentiation and bone regeneration is characterized by comprising the following raw materials: graphene oxide, polyethyleneimine, micro RNA-214 inhibitor, silk fibroin and nano hydroxyapatite;
the preparation method of the micro RNA-214 inhibitor loaded degradable material with the functions of inducing osteogenic differentiation and bone regeneration comprises the following steps:
(1) preparing graphene oxide and polyethyleneimine into a solution with a certain concentration, mixing, performing ultrasonic treatment, and stirring to obtain a graphene oxide-polyethyleneimine compound solution;
(2) compounding the compound solution obtained in the step (1) with a micro RNA-214 inhibitor in a phosphate buffer solution, vortexing at room temperature, and incubating to obtain a graphene oxide-polyethyleneimine compound solution loaded with the micro RNA-214 inhibitor;
(3) mixing and stirring an ammonium hydrogen phosphate solution and a calcium chloride solution, aging and centrifuging at room temperature, and freeze-drying the obtained precipitate to obtain nano-hydroxyapatite particles; drying the silk fibroin extracted from the silkworm cocoon, placing the dried silk fibroin in a lithium bromide solution, heating and dissolving to obtain a silk fibroin solution, dialyzing, and centrifugally collecting for later use;
(4) mixing and stirring the silk fibroin solution obtained in the step (3), nano hydroxyapatite and the oxidized graphene-polyethyleneimine loaded micro-RNA-214 inhibitor compound obtained in the step (2), adding the mixture into a pore plate, and freeze-drying to obtain a degradable material with osteogenic differentiation and bone regeneration induction functions;
the concentration of the graphene oxide in the step (1) is 0.1-10 mg/mL; the concentration of the polyethyleneimine is 0.1-10 mg/mL; wherein the mass ratio of the graphene oxide to the polyethyleneimine in the reaction system is 1: 1-5; the mass ratio of the graphene oxide-polyethyleneimine composite to the micro RNA-214 inhibitor in the step (2) is 100-30: 1.
2. The micro RNA-214 inhibitor-loaded degradable material with the osteogenic differentiation and bone regeneration inducing function of claim 1, wherein the concentration of the graphene oxide in the step (1) is 1 mg/mL; the concentration of polyethyleneimine is 1 mg/mL; the mass ratio of the graphene oxide to the polyethyleneimine in the reaction system is 1: 2.
3. The micro RNA-214 inhibitor-loaded degradable material with the function of inducing osteogenic differentiation and bone regeneration according to claim 1, wherein the ratio of the graphene oxide-polyethyleneimine complex to the micro RNA-214 inhibitor in the step (2) is 30: 1.
4. The micro RNA-214 inhibitor-loaded degradable material with the osteogenic differentiation and bone regeneration inducing function of claim 1, wherein the concentration of ammonium hydrogen phosphate in the reaction system in the step (3) is 0.1-1 mol/L; the concentration of calcium chloride in the reaction system is 0.1-1 mol/L; the concentration of lithium bromide in the reaction system is 1-20 mol/L; the mass fraction of the silk fibroin in the reaction system is 1-10%.
5. The micro RNA-214 inhibitor-loaded degradable material with the osteogenic differentiation and bone regeneration inducing function of claim 1, wherein the concentration of the graphene oxide-polyethyleneimine-micro RNA-214 inhibitor in the system in the step (4) is 0.1-10 mg/mL.
6. Use of the micro RNA-214 inhibitor loaded degradable material with osteogenic differentiation and bone regeneration inducing function of claim 1 in preparation of bone repair materials.
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