CN110180029A - A kind of preparation method and application with induced osteogenesis differentiation and the degradation material of bone regeneration function - Google Patents
A kind of preparation method and application with induced osteogenesis differentiation and the degradation material of bone regeneration function Download PDFInfo
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Abstract
The present invention discloses a kind of load micro RNA-214 inhibitor degradation material with induced osteogenesis differentiation and bone regeneration function, is made of following raw material: graphene oxide, polyethyleneimine, micro RNA-214 inhibitor, fibroin albumen, nanometer hydroxyapatite.Of the invention has induced osteogenesis differentiation degradable in vivo with the load micro RNA-214 inhibitor degradation material of bone regeneration function, have the function of preferable Osteoblast Differentiation and higher bioactivity, promoting cell proliferation and differentiation, cell adherence etc. is promoted to have a clear superiority, and catabolite has no significant effect ambient enviroment.Therefore, minimally invasive Bone Defect Repari application of the load microRNA-214 inhibitor degradation material with induced osteogenesis differentiation and bone regeneration function of the invention suitable for clinic.
Description
Technical field
The present invention relates to biomedical material technologies, and in particular to one kind has induced osteogenesis differentiation and osteanagenesis function
The preparation method and application of the degradation material of energy.
Background technique
Currently, bone defect has become for most common disease clinically.For patient biggish for bone defect area, by
To the factors limitation such as destruction or repairing environment, regeneration and repair ability by bone itself, it tends to be difficult to reach self-healing effect.It replaces
It is the common method of large area bone defect healing for material, and has broad application prospects.Bone tissue engineer is that one kind has very much
The method of future, it is designed for because of traffic accident, traumatic damage, skeletal diseases, tumor resection, bone necrosis or wind
The bone substitute of repair of fractured bones caused by diseases caused by dampness/regeneration new bone tissue provides suitable technology.But bone substitute/transplanting
Object (of the same race/self) due to lack ideal bone substitute, the osteogenic potential of graft, immunological rejection and secondary sexual trauma and by
To limitation.Micro RNA-214 can target the transcription factor ATF4 in osteoblast and inhibit bon e formation, micro RNA-214
Inhibitor has the activity for promoting skeletonization, inhibits the dual regulation of bone active, the AKT and ERK1/2 of osteoblast is activated to believe
Number access, to promote the differentiation of cell.For these problems, we develop a kind of load micro RNA-214 inhibitor
With induced osteogenesis differentiation and the degradable three-dimensional rack of bone regeneration function.The present invention is in terms of Osteoblast Differentiation and osteanagenesis two
Degradation material with Bone Defect Repari function is analyzed.The result shows that the tool of load micro RNA-214 inhibitor
There are induced osteogenesis differentiation and the degradable three-dimensional rack of bone regeneration function that can not only promote the adherency of cell that can also effectively promote
Into the differentiation and osteanagenesis of skeletonization.
It is mainly clinically bone collection and bone alternate material for the common treatment means of bone defect.Wherein bone collection includes
Autologous bone, allograph bone and bone xenograft.Although autologous bone transplanting will not generate immunological rejection but also have good
Self-bone grafting and osteoconductive energy, but its source is extremely limited；Although the source of allograph bone and bone- xenograft is abundant, can generate immune
Rejection, can also with infection etc. complication and also bone inductive effect it is poor.
Summary of the invention
It is prepared a kind of with induced osteogenesis differentiation it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
With the load micro RNA-214 inhibitor degradation material of bone regeneration function, this kind of material can promote cell adherence and at
Bone differentiation and bon e formation, are better than traditional bone collection and traditional bracket, and its degradation speed and Bone Defect Repari rate phase
Match.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of load micro RNA-214 inhibitor degradation material with induced osteogenesis differentiation and bone regeneration function,
It is characterized in that, being made of following raw material: graphene oxide, polyethyleneimine, micro RNA-214 inhibitor, fibroin albumen,
Nanometer hydroxyapatite.
The load micro RNA-214 inhibition with induced osteogenesis differentiation with bone regeneration function that the present invention also provides a kind of
The raw preparation method of agent degradation material, comprising the following steps:
(1) it mixes after graphene oxide and polyethyleneimine being configured to certain density solution, is then ultrasonically treated, stir
It mixes, obtains graphene oxide-polyethyleneimine complex solution；
(2) complex solution of step (1) and micro RNA-214 inhibitor is compound in phosphate buffer, room
It is vortexed under the conditions of temperature, after incubation, obtains the graphene oxide-polyethyleneimine amine compound for being loaded with micro RNA-214 inhibitor
Solution；
It (3), will be resulting after room temperature condition ageing centrifugation after ammonium hydrogen phosphate solution and calcium chloride solution being mixed
Sediment freeze-drying, obtains nano-hydroapatite particles；Again by after the fibroin drying extracted in silk cocoon, it is placed in lithium bromide
In solution, after heating for dissolving, silk fibroin protein solution is obtained, after dialysing, is collected by centrifugation, it is spare；
(4) by the graphite oxide of silk fibroin protein solution and nanometer hydroxyapatite and step (2) that concentration is step (3)
After alkene-polyethyleneimine load micro-RNA-214 inhibitor complexes are mixed, it is added in orifice plate, is obtained after freeze-drying
Degradation material with induced osteogenesis differentiation and bone regeneration function.
Preferably, the concentration of graphene oxide is 0.1~10mg/mL in the step (1)；The polyethyleneimine it is dense
Degree is 0.1~10mg/mL.
Preferably, the concentration of graphene oxide is 1mg/mL in the step (1)；The concentration of polyethyleneimine is 1mg/
mL。
Preferably, in the step (1) mass ratio of graphene oxide and polyethyleneimine in the reaction system be 1:1~
5。
Preferably, the mass ratio of graphene oxide and polyethyleneimine in the reaction system is 1:2 in the step (1).
Preferably, ultrasonic time is 10~60min in the step (1)；Mixing time be 3~for 24 hours.
Preferably, ultrasonic time is 15min in the step (1)；Mixing time is 12h.
Preferably, graphene oxide-polyethyleneimine amine compound described in the step (2) and micro RNA-214
The mass ratio of inhibitor is 100~30:1.
Preferably, graphene oxide-polyethyleneimine amine compound described in the step (2) and micro RNA-214
The mass ratio of inhibitor is 30:1.
Preferably, vortex time described in the step (2) is 30~100s；The incubation time is described incubates
Educating the time is 10~120min.
Preferably, vortex time described in the step (2) is 60~100s；The incubation time is described incubates
Educating the time is 60min.
Preferably, the concentration of ammonium hydrogen phosphate in the reaction system is 0.1~1mol/L in the step (3)；Calcium chloride exists
Concentration in reaction system is 0.1~1mol/L；The concentration of lithium bromide in the reaction system is 1~20mol/L；Fibroin albumen exists
Mass fraction in reaction system is 1~10%.
Preferably, the concentration of ammonium hydrogen phosphate in the reaction system is 0.3mol/L in the step (3)；Calcium chloride is reacting
Concentration in system is 0.5mol/L；The concentration of lithium bromide in the reaction system is 9.3mol/L；Fibroin albumen is in reaction system
In mass fraction be 4%.
Preferably, the time stirred in the step (3) is 1~6h；12~36h between ageing centrifugation；Centrifugal rotational speed is
1000~10000rpm, centrifugation time are 5~60min；Heating time is 1~12h, and heating temperature is 30~100 DEG C.
Preferably, the time stirred in the step (3) is 2h；Between ageing centrifugation for 24 hours；Centrifugal rotational speed is 5000rpm, from
The heart time is 10min；Heating time is 5h, and heating temperature is 60 DEG C.
Preferably, graphene oxide described in the step (4)-polyethyleneimine-micro RNA-214 inhibitor is multiple
Closing concentration of the object in system is 0.1~10mg/mL.
Preferably, graphene oxide described in the step (4)-polyethyleneimine-micro RNA-214 inhibitor is multiple
Closing concentration of the object in system is 1mg/mL.
It is a kind of as described above with induced osteogenesis differentiation and bone regeneration function another object of the present invention is to also provide
Application of the load micro RNA-214 inhibitor degradation material in Bone Defect Repari.
Beneficial effects of the present invention: the present invention provides a kind of loads with induced osteogenesis differentiation and bone regeneration function
Micro RNA-214 inhibitor degradation material, this kind of degradation material are made of nanometer hydroxyapatite and fibroin albumen
Bracket and polyethyleneimine it is amine-modified graphene oxide-loaded micro RNA-214 inhibitor complexes composition.Of the invention
It is degradable in vivo with the load micro RNA-214 inhibitor degradation material of bone regeneration function with induced osteogenesis differentiation,
Have the function of preferable Osteoblast Differentiation and higher bioactivity, is promoting cell proliferation and differentiation, promoting cell adherence etc.
It has a clear superiority, and catabolite has no significant effect ambient enviroment.Therefore, kind of the invention has induced osteogenesis differentiation and bone
Minimally invasive Bone Defect Repari application of the load microRNA-214 inhibitor degradation material of regeneration function suitable for clinic.
Detailed description of the invention
Fig. 1 is the infrared spectrogram of graphene oxide, polyethyleneimine and polyethyleneimine-modified graphene oxide.
Fig. 2 is the transmission electron microscope picture of graphene oxide and polyethyleneimine-modified graphene oxide.
Fig. 3 is the gel retardation assasy result that the present invention has induced osteogenesis differentiation and the degradation material of bone regeneration function
Schematic diagram.
Fig. 4 is the cell activity schematic diagram that the present invention has induced osteogenesis differentiation and the degradation material of bone regeneration function.
Fig. 5 is that the present invention has induced osteogenesis differentiation and the degradation material alkaline phosphatase (ALP) of bone regeneration function living
Property schematic diagram.
Specific embodiment
For more concise displaying technical solution of the present invention, objects and advantages, combined with specific embodiments below
And its attached drawing is described in further detail the present invention.
The preparation method of the present invention load micro RNA-214 inhibitor degradation material of embodiment 1 the following steps are included:
Step 1: the synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene oxide (GO) is configured to spare after 1mg/mL ultrasonic treatment, then polyethyleneimine (25k) (PEI) is matched
It is set to 1mg/mL solution and is slowly added into 10min in GO solution.By the way that GO solution and diluted PEI solution are pressed 1:
2 mass ratio mixing after being then ultrasonically treated 15min, is stirred 12 hours overnight, is then removed by the method for centrifugation and washing
Free PEI obtains GO-PEI complex solution.
Step 2: graphene oxide-polyethyleneimine loads micro-RNA-214 inhibitor complexes (GO-PEI-
MicroRNA-214 synthesis)
By GO-PEI complex solution and micro RNA-214 inhibitor with the mass ratio of 30:1 in phosphate buffer
Compound, be vortexed 60s under room temperature, is incubated for 60min to get the poly- second of graphene oxide-for being loaded with micro RNA-214 inhibitor
Alkene imines compound.Wherein: the specifying information of micro RNA-214 inhibitor is as follows: miRNA title: mmu-miR-214-3p
MiRBase Accession:MIMAT0000661 miRNA maturation body sequence: acagcaggcacagacaggcagu.
Step 3: the preparation of fibroin albumen and nanometer hydroxyapatite
After the calcium chloride solution that the ammonium hydrogen phosphate solution that concentration is 0.3mol/L is 0.5mol/L with concentration is mixed,
At room temperature condition (pH=10) ageing centrifugation 24 hours, resulting sediment was freeze-dried 24 hours, obtains nano-hydroxy-apatite
Stone particle.Again by after the fibroin drying extracted in silk cocoon, it is placed in the lithium-bromide solution that concentration is 9.3mol/mL, 60 DEG C add
After heat of solution 5h, silk fibroin protein solution is obtained, then dialyse 3d, and 5000rpm is centrifuged 10min and collects, spare.
Step 4: the synthesis with induced osteogenesis differentiation and the degradation material of bone regeneration function
It is 1mg/mL's by the silk fibroin protein solution 1mL of concentration 4% (w/v) and 10mg nanometer hydroxyapatite and concentration
After graphene oxide-polyethyleneimine load micro-RNA-214 inhibitor complexes solution 1mL is mixed, it is added to 24
The degradation material with induced osteogenesis differentiation and bone regeneration function is obtained in orifice plate, after freeze-drying.
The preparation method of the present invention load micro RNA-214 inhibitor degradation material of embodiment 2 the following steps are included:
Step 1: the synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene oxide (GO) is configured to it is spare after 0.1mg/mL ultrasonic treatment, then by polyethyleneimine (25k) (PEI)
It is configured to 0.1mg/mL solution and is slowly added into 10min in GO solution.By by GO solution and diluted PEI solution
It is mixed by the mass ratio of 1:1, after being then ultrasonically treated 15min, stirs 12 hours overnight, then pass through the method for centrifugation and washing
Free PEI is removed, GO-PEI complex solution is obtained.
Step 2: graphene oxide-polyethyleneimine loads micro-RNA-214 inhibitor complexes (GO-PEI-
MicroRNA-214 synthesis)
By GO-PEI complex solution and micro RNA-214 inhibitor with the quality of 100:1 in phosphate buffer
Than compound, be vortexed 60s under room temperature, and the graphene oxide-for being incubated for 60min to get micro RNA-214 inhibitor is loaded with is poly-
Aziridine compound.
Step 3: the preparation of fibroin albumen and nanometer hydroxyapatite
After the calcium chloride solution that the ammonium hydrogen phosphate solution that concentration is 0.1mol/L is 0.1mol/L with concentration is mixed,
At room temperature condition (pH=10) ageing centrifugation 24 hours, resulting sediment was freeze-dried 24 hours, obtains nano-hydroxy-apatite
Stone particle.Again by after the fibroin drying extracted in silk cocoon, it is placed in the lithium-bromide solution that concentration is 1mol/mL, 60 DEG C of heating
After dissolving 5h, silk fibroin protein solution is obtained, then dialyse 3d, and 5000rpm is centrifuged 10min and collects, spare.
Step 4: the synthesis with induced osteogenesis differentiation and the degradation material of bone regeneration function
It is 0.1mg/ by silk fibroin protein solution 1mL and 10mg nanometer hydroxyapatite and concentration that concentration is 1% (w/v)
After the graphene oxide of mL-polyethyleneimine load micro-RNA-214 inhibitor complexes solution 1mL is mixed, it is added
The degradation material with induced osteogenesis differentiation and bone regeneration function is obtained into 24 orifice plates, after freeze-drying.
The preparation method of the present invention load micro RNA-214 inhibitor degradation material of embodiment 3 the following steps are included:
Step 1: the synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene oxide (GO) is configured to spare after 6mg/mL ultrasonic treatment, then polyethyleneimine (25k) (PEI) is matched
It is set to 6mg/mL solution and is slowly added into 10min in GO solution.By the way that GO solution and diluted PEI solution are pressed 1:
4 mass ratio mixing after being then ultrasonically treated 15min, is stirred 12 hours overnight, is then removed by the method for centrifugation and washing
Free PEI obtains GO-PEI complex solution.
Step 2: graphene oxide-polyethyleneimine loads micro-RNA-214 inhibitor complexes (GO-PEI-
MicroRNA-214 synthesis)
By GO-PEI complex solution and micro RNA-214 inhibitor with the mass ratio of 60:1 in phosphate buffer
Compound, be vortexed 60s under room temperature, is incubated for 60min to get the poly- second of graphene oxide-for being loaded with micro RNA-214 inhibitor
Alkene imines compound.
Step 3: the preparation of fibroin albumen and nanometer hydroxyapatite
After the calcium chloride solution that the ammonium hydrogen phosphate solution that concentration is 0.6mol/L is 0.8mol/L with concentration is mixed,
At room temperature condition (pH=10) ageing centrifugation 24 hours, resulting sediment was freeze-dried 24 hours, obtains nano-hydroxy-apatite
Stone particle.Again by after the fibroin drying extracted in silk cocoon, it is placed in the lithium-bromide solution that concentration is 5mol/mL, 60 DEG C of heating
After dissolving 5h, silk fibroin protein solution is obtained, then dialyse 3d, and 5000rpm is centrifuged 10min and collects, spare.
Step 4: the synthesis with induced osteogenesis differentiation and the degradation material of bone regeneration function
It is 5mg/mL by silk fibroin protein solution 1mL and 10mg nanometer hydroxyapatite and concentration that concentration is 8% (w/v)
Graphene oxide-polyethyleneimine load micro-RNA-214 inhibitor complexes solution 1mL be mixed after, be added to
The degradation material with induced osteogenesis differentiation and bone regeneration function is obtained in 24 orifice plates, after freeze-drying.
The preparation method of the present invention load micro RNA-214 inhibitor degradation material of embodiment 4 the following steps are included:
Step 1: the synthesis of graphene oxide-polyethyleneimine (GO-PEI)
Graphene oxide (GO) is configured to it is spare after 10mg/mL ultrasonic treatment, then by polyethyleneimine (25k) (PEI)
It is configured to 10mg/mL solution and is slowly added into 10min in GO solution.By by GO solution and diluted PEI solution
It is mixed by the mass ratio of 1:5, after being then ultrasonically treated 15min, stirs 12 hours overnight, then pass through the method for centrifugation and washing
Free PEI is removed, GO-PEI complex solution is obtained.
Step 2: graphene oxide-polyethyleneimine loads micro-RNA-214 inhibitor complexes (GO-PEI-
MicroRNA-214 synthesis)
By GO-PEI complex solution and micro RNA-214 inhibitor with the mass ratio of 90:1 in phosphate buffer
Compound, be vortexed 60s under room temperature, is incubated for 60min to get the poly- second of graphene oxide-for being loaded with micro RNA-214 inhibitor
Alkene imines compound.
Step 3: the preparation of fibroin albumen and nanometer hydroxyapatite
After the calcium chloride solution that the ammonium hydrogen phosphate solution that concentration is 1mol/L is 1mol/L with concentration is mixed, in room
Warm condition (pH=10) ageing centrifugation 24 hours, resulting sediment is freeze-dried 24 hours, obtains nanometer hydroxyapatite
Grain.Again by after the fibroin drying extracted in silk cocoon, it is placed in the lithium-bromide solution that concentration is 20mol/mL, 60 DEG C of heating for dissolving
After 5h, silk fibroin protein solution is obtained, then dialyse 3d, and 5000rpm is centrifuged 10min and collects, spare.
Step 4: the synthesis with induced osteogenesis differentiation and the degradation material of bone regeneration function
It is 10mg/ by silk fibroin protein solution 1mL and 10mg nanometer hydroxyapatite and concentration that concentration is 10% (w/v)
After the graphene oxide of mL-polyethyleneimine load micro-RNA-214 inhibitor complexes solution 1mL is mixed, it is added
The degradation material with induced osteogenesis differentiation and bone regeneration function is obtained into 24 orifice plates, after freeze-drying.
Embodiment 5: the synthesis of graphene oxide-polyethyleneimine (GO-PEI) and infrared analysis
FTIR Fourier infrared spectrum is a kind of spectrum for showing molecular vibration, can identify the functional group in test substance,
The variation of GO, PEI, GO-PEI characteristic peak is characterized by Fourier infrared spectrum.GO-PEI and raw material GO, PEI are done into infrared test,
Whether provable target product is successfully synthesized.GO, PEI of 1-2mg and the sample of the GO-PEI of embodiment 1 is taken to be placed in Ma respectively
After Nao mortar is ground, (sample to be tested is in potassium bromide for the potassium bromide powder after sequentially adding 100-200mg drying 24 hours
Ratio is about 0.5%-1%) to continue to be ground to granularity careful, after mixing, by mixture at vacuum degree 10mmHg 2-
5min is pressed into transparent sheet and is tested, scanning range 4000-400cm-1, it is as shown in Figure 1 to obtain result.Fourier transformation is red
Outside (FT-IR) analysis shows, GO and GO-PEI all have-OH, C=O and C-O group, correspond respectively to 3200-3500cm-1、
1500-1760cm-1And 1000-1500cm-1Peak value.C-H, N-H and C-N vibration peak of PEI and GO-PEI corresponds respectively to
500-900cm-1、1000-1250cm-1And 1250-1500cm-1.These peaks the result shows that PEI is to pass through electrostatic interaction
And non-covalent bond is grafted on graphene oxide, it was demonstrated that the GO-PEI compound in embodiment 1 successfully synthesizes.
Embodiment 6: the synthesis of graphene oxide-polyethyleneimine (GO-PEI) and the analysis of transmission electron microscope
The microscopic appearance of the GO-PEI composite particles of 1 step 1 of embodiment preparation is observed by transmission electron microscope (TEM).
Show that surface of graphene oxide relative smooth, the graphene oxide of PEI functionalization change oxidation from Fig. 2 transmission electron microscope analysis
The smoothness of graphene surface increases the size of graphene oxide.
The gel blocking of embodiment 7:GO-PEI and micro RNA-214 inhibitor
It is female that micro RNA-214 inhibitor is rapidly added to the resulting graphene oxide of embodiment 1-polyethyleneimine
It is slowly 8~10 times, 25 DEG C of incubation 30min reverse up and down in liquid, for use.It is prepared for a series of different quality ratio (GO-PEI/
Micro RNA-214) nano combined compound, the mass ratio set is 0,10,20,30,40,50 and 60.It is every by changing
The additional amount of group GO-PEI, to ensure that the additional amount of each group micro RNA-214 inhibitor is consistent, for use in subsequent solidifying
The detection of glue retardation experiment.1% Ago-Gel for preparing the dye liquor containing 0.1%EB again, is gently added above-mentioned preparation in duct
Good each group sample submerges gel with TAE buffer.Voltage 100V is set, starts to carry out electrophoresis, after 25min, stops electrophoresis.
Ago-Gel is carefully taken out, after ultraviolet irradiation, is taken pictures with gel imaging system to it, as a result as shown in Figure 3.It is real
Test the result shows that, when the mass ratio of GO-PEI and micro RNA-214 inhibitor is less than or equal to 20, micro RNA-214 suppression
Preparation is not completely combined with GO-PEI, when the two mass ratio reaches 30, is arrested at loading hole, and the two, which is formed, closely to be received
Rice composite structure.Therefore selecting mass ratio in subsequent experimental is 30 GO-PEI/micro RNA-214.
Embodiment 8: the degradable material of load microvRNA-214 inhibitor with induced osteogenesis differentiation and bone regeneration function
The cytoactive detection of the system of material
Degradation material (SF/HAP/GPM) the progress cytoactive detection by CCK-8 method prepared by embodiment 1, and with
Silk fibroin bracket material (SF) and fibroin albumen-nanometer hydroxyapatite compound rest (SF/HAP) are to compare.This experiment
Cell used is fibroblast (3T3 cell), and cultivating culture solution used in the cell is the fetal calf serum containing 10%
With the culture solution of the DMEM of 1% dual anti-(mixed liquor of penicillin and streptomysin), and condition of culture be temperature be 37 DEG C
And CO2In the incubator that concentration is 5%.During culture, a culture solution is changed to cell within every two days, change cell culture
The purpose of liquid is the metabolin for increasing new nutriment for cell, removing not adherent cell and cell.By what is sterilized
The material of difference group is placed in 48 orifice plates, is then added drop-wise to the cell suspending liquid of the 50 μ L adjusted and is broken up with induced osteogenesis
In the load microRNA-214 inhibitor degradation material of bone regeneration function, in the incubator, after being incubated for 2h, 450 μ L are taken
Culture solution be added on material, continue to cultivate.CCK-8 reagent is added after cultivating by 3 and 7 days respectively, according to the ratio of 1:10
It is added, that is to say, that the CCK-8 reagent of 10 μ L is added in the culture solution of 100 μ L, continues to cultivate 2-4h.In the condition of 450nm wavelength
Under, the absorption photometric value in each hole is read using microplate reader.As a result as shown in Figure 4, the results showed that: SF/HAP/GPM bracket it is thin
Born of the same parents' proliferation rate is above SF and SF/HAP bracket.3rd day, cell Proliferation reached higher level；However, at the 7th day, the increasing of cell
Reduction is grown, cell tends to differentiation and mineralising.The above result shows that of the invention has induced osteogenesis differentiation and bone regeneration function
Load microRNA-214 inhibitor degradation material have good biocompatibility and cell adhesion.
Embodiment 9: the degradable material of load microRNA-214 inhibitor with induced osteogenesis differentiation and bone regeneration function
Alkaline phosphatase (ALP) Activity determination of material
Have induced osteogenesis differentiation degradable with the load microRNA-214 inhibitor of bone regeneration function for sterilized
Material (SF/HAP/GPM) and silk fibroin bracket material (SF) and fibroin albumen-hydroxyapatite scaffold material (SF/HAP) divide
It is not placed in 48 well culture plates.Culture is taken to digest it from culture bottle using 0.25% pancreatin to the cell in 3 generations,
It is again that 1000rpm is centrifuged with revolving speed, time 5min discards supernatant liquid, then will contain serum and dual anti-(penicillin and chain
Mixed liquor) α-DMEM culture solution be added thereto, and adjusting cell concentration is every milliliter containing 5 × 107A cell.Each sample
The above-mentioned cell suspending liquid that 20 μ L are planted on product, places it in 37 DEG C, 5%CO2Incubator in be incubated for 2 in the incubator that is incubated for
A hour adds the culture of culture solution continuation of 500 μ L to 7 days, 14 days and 21 days.In the training period, replacement in 2-3 days is primary
Culture solution, in order to which cell can obtain enough nutrition.Material is taken out from orifice plate, then will with sterile PBS solution
With induced osteogenesis differentiation and load microRNA-214 inhibitor degradation material rinse 3 times of bone regeneration function, then will
500 μ L cell pyrolysis liquids are added thereto, and are then placed on carrying out clasmatosis in the ultrasonic cell disintegration instrument that temperature is 4 DEG C.It is right
It is centrifuged, and collects supernatant.500 μ L ALP substrate reactions liquids are added in supernatant, under conditions of bath temperature is 37 DEG C
30min is reacted, in order to terminate reaction, the concentration that 500 μ L are added into reaction solution is the NaOH of 0.1M, then uses ultraviolet-visible
Protractor measures light splitting angle value of the sample at 405nm, calculates ALP by specification.Every group of material of each time point is at least
Parallel testing 3 times.Experimental result is as shown in figure 5, break up the load with bone regeneration function with induced osteogenesis in 14 days
The ALP activity of cell in microRNA-214 inhibitor degradation material shows increased with the extension of incubation time
Trend, the results showed that, the load microRNA-214 inhibitor degradation material with induced osteogenesis differentiation and bone regeneration function
Be conducive to the differentiation of skeletonization, and show the Osteoblast Differentiation ability better than the material that microRNA-214 inhibitor is not added.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (9)
1. a kind of load micro RNA-214 inhibitor degradation material with induced osteogenesis differentiation and bone regeneration function,
Be characterized in that, be made of following raw material: graphene oxide, micro RNA-214 inhibitor, fibroin albumen, is received polyethyleneimine
Rice hydroxyapatite.
2. a kind of press down with induced osteogenesis differentiation and the load micro RNA-214 of bone regeneration function as described in claim 1
The raw preparation method of preparation degradation material, which comprises the following steps:
(1) it mixes after graphene oxide and polyethyleneimine being configured to certain density solution, is then ultrasonically treated, stir,
Obtain graphene oxide-polyethyleneimine complex solution；
(2) complex solution of step (1) and micro RNA-214 inhibitor is compound in phosphate buffer, room temperature item
It is vortexed under part, after incubation, obtains the graphene oxide-polyethyleneimine complex solution for being loaded with micro RNA-214 inhibitor；
(3) after ammonium hydrogen phosphate solution and calcium chloride solution being mixed, after room temperature condition ageing centrifugation, by resulting precipitating
Object freeze-drying, obtains nano-hydroapatite particles；Again by after the fibroin drying extracted in silk cocoon, it is placed in lithium-bromide solution
In, after heating for dissolving, silk fibroin protein solution is obtained, after dialysing, is collected by centrifugation, it is spare；
(4) by the silk fibroin protein solution of step (3) and nanometer hydroxyapatite and graphene oxide-polyethylene of step (2)
Imines load micro-RNA-214 inhibitor complexes be mixed after, be added in orifice plate, obtained after freeze-drying have induction at
The degradation material of bone differentiation and bone regeneration function.
3. preparation method as claimed in claim 2, which is characterized in that the concentration of graphene oxide is 0.1 in the step (1)
~10mg/mL；The concentration of the polyethyleneimine is 0.1~10mg/mL；Wherein graphene oxide is being reacted with polyethyleneimine
Mass ratio in system is 1:1~5.
4. preparation method as claimed in claim 2, which is characterized in that the concentration of graphene oxide is in the step (1)
1mg/mL；The concentration of polyethyleneimine is 1mg/mL；Graphene oxide is with the mass ratio of polyethyleneimine in the reaction system
1:2。
5. preparation method as claimed in claim 2, which is characterized in that the poly- second of graphene oxide-described in the step (2)
The mass ratio of alkene imines compound and micro RNA-214 inhibitor is 100~30:1.
6. preparation method as claimed in claim 2, which is characterized in that the poly- second of graphene oxide-described in the step (2)
The ratio of alkene imines compound and micro RNA-214 inhibitor is 30:1.
7. preparation method as claimed in claim 2, which is characterized in that ammonium hydrogen phosphate is in the reaction system in the step (3)
Concentration be 0.1~1mol/L；The concentration of calcium chloride in the reaction system is 0.1~1mol/L；Lithium bromide is in the reaction system
Concentration be 1~20mol/L；The mass fraction of fibroin albumen in the reaction system is 1~10%.
8. preparation method as claimed in claim 2, which is characterized in that the poly- second of graphene oxide-described in the step (4)
Concentration of the alkene imines-micro RNA-214 inhibitor in system is 0.1~10mg/mL.
9. a kind of press down with induced osteogenesis differentiation and the load micro RNA-214 of bone regeneration function as described in claim 1
Application of the preparation degradation material in Bone Defect Repari.
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