CN104225685B - A kind of preparation method conducting electricity spacetabs type neural tissue engineering support - Google Patents
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support Download PDFInfo
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- CN104225685B CN104225685B CN201410478759.0A CN201410478759A CN104225685B CN 104225685 B CN104225685 B CN 104225685B CN 201410478759 A CN201410478759 A CN 201410478759A CN 104225685 B CN104225685 B CN 104225685B
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Abstract
The present invention relates to a kind of preparation method conducting electricity spacetabs type neural tissue engineering support, including: fibroin albumen Silk and lactic acid caprolactone copolymer are dissolved in solvent, dissolve stirring, obtain solution, it is subsequently adding polyaniline PANi and camphorsulfonic acid, stirs and evenly mixs, obtain cortex electrostatic spinning solution；Nerve growth factor is dissolved completely in ultra-pure water, obtains sandwich layer electrostatic spinning solution；Cortex electrostatic spinning solution, sandwich layer electrostatic spinning solution are respectively charged in syringe, carry out coaxial electrostatic spinning, then suffocating treatment, vacuum drying, obtain conductivity type neural tissue engineering support.The approach such as the nano fiber scaffold that the present invention prepares topological structure orientation guide needed for additional electro photoluminescence conducting polymer, biochemistry nerve growth factor and nerve regneration promote CO2 laser weld speed.The inventive method is simple to operate, favorable repeatability, high financial profit, and the neurologic defect reparation for running in clinic provides new experimental considerations.
Description
Technical field
The invention belongs to neurologic defect reparation and relate to biomaterial preparation field, prop up particularly to one conduction spacetabs type neural tissue engineering
The preparation method of frame.
Background technology
Perineural regeneration and the difficult problem recovering always puzzlement clinic of function, there is substantial amounts of trouble in the annual whole world
Person needs to carry out CO2 laser weld.Up to the present, the method being most frequently with is that nerve autograft is to treat neurotmesis or to lack
Damage.But, the source that autologous nerve is available for transplanting is very limited.This external microsurgery is difficult to avoid nerve fibre
Misorientation docks, thus affects the recovery of nervous function.Recently as the progressively development of organizational project, biomaterial is utilized to build
Nerve regeneration conduit, bring hope for clinical neurologic defect reparation.
But peripheral nerve regeneration not only to recover its structure, it is often more important that recover its domination function to target organ, in order to reach
To or exceed the repairing effect of nerve autograft, Recent study persons constantly endeavour controllable activity composite nerve conduit
Preparation.It is known that nerve conduction be the change of film potential on nerve fibre at all, nerve conduction is the biography of the signal of telecommunication
Lead, have the report electro photoluminescence of a lot of document can promote and guide nerve cell migration in recent years.Meanwhile, nerve growth factor
Motorial regeneration can also be played a driving role by (nerve growth factor, NGF), to so far, there are no based on
The report of coaxial electrostatic spinning technology preparation one load nerve growth factor (NGF) conductivity type neural tissue engineering support.
Summary of the invention
The technical problem to be solved is to provide a kind of preparation method conducting electricity spacetabs type neural tissue engineering support, this
Bright advantage is to utilize cleverly coaxial electrostatic spinning technology, obtains conduction spacetabs type nano-fiber nerve conduit, its biofacies
Capacitive is good, it is possible to guides nerve cell along oriented growth, the extension of accelerans aixs cylinder, promotes injured nerve Regeneration and Repair；
This method is simple to operate, favorable repeatability, economic benefit.
The present invention a kind of conducts electricity the preparation method of spacetabs type neural tissue engineering support, including:
(1) fibroin albumen and lactic acid-caprol acton copolymer that mass ratio is 25-50:75-50 are dissolved in solvent, dissolve stirring,
It is the solution of 8-10% to quality percent by volume, is subsequently adding polyaniline PANi and camphorsulfonic acid that mass ratio is 1:1,
Stir and evenly mix, obtain cortex electrostatic spinning solution；
(2) nerve growth factor is dissolved completely in ultra-pure water, obtains sandwich layer electrostatic spinning solution；
(3) cortex electrostatic spinning solution, sandwich layer electrostatic spinning solution are respectively charged in syringe, carry out coaxial electrostatic spinning, so
Rear suffocating treatment, vacuum drying, must conduct electricity spacetabs type neural tissue engineering support.
In described step (1), the molecular weight of lactic acid-caprol acton copolymer is Mn≈ 300,000, the ratio of lactic acid-caprol acton is 50:50, poly-
The molecular weight Mn ≈ 1.5 ten thousand of aniline PANi.
In described step (1), solvent is hexafluoroisopropanol.
In described step (1), dissolved stirring time is 5-6h, adds polyaniline PANi and camphorsulfonic acid, and the time of stirring is 24-48h,
Stir speed (S.S.) is 200-300r/min.
In described step (1), camphorsulfonic acid can make polyaniline have conductive characteristic.
In described step (1) mediopellis electrostatic spinning solution, the quality percent by volume of polyaniline PANi is 1.2-1.6%.
In described step (2), the raw sub-factor (NGF) solution of the nerve of ultra-pure water preparation needs now with the current, it is to avoid place impact for a long time
Its activity.
In described step (2) center core layer electrostatic spinning solution, the concentration of nerve growth factor is 10 μ g/ml.
Coaxial electrostatic spinning technique in described step (3), particularly as follows: cortex electrostatic spinning solution loads in syringe, advances pump at trace
Effect lower enter at the annular spaces that coaxial device is combined between the inside and outside capillary spinning nozzle of capillary shower nozzle；By sandwich layer electrostatic spinning
Solution loads syringe, and the dynamic lower entrance coaxial device of effect at micro-injection pump is combined the interior capillary spinning head of capillary shower nozzle, opens
Open electrostatic pressure generator, advance pump, and cylinder receives the motor of device, carries out spinning.
Described spinning nozzle is the composite capillary of concentric shafts, and prepared nanofiber has " core-shell " structure.
In described step (3), coaxial electrostatic spinning technological parameter is: spinning nozzle be internal layer be 8 extra floor be No. 16 concentric shafts spray
Silk head；The fltting speed of syringe pump is respectively sandwich layer 0.2ml/h and shell 1.0ml/h；Dress is received at concentric shafts spinning head and cylinder
The regulation voltage accessed between putting is 12-13kV；The distance that concentric shafts spinning head and cylinder receive between device is 5-6cm；Rolling
It is 4000r/min that cylinder motor is supplied to the rotating speed of cylinder.
In described step (3), suffocating treatment is that ethanol carries out suffocating treatment.
The percentage by volume of described ethanol is 75%, and the suffocating treatment time is 24-48h.
In described step (3), the vacuum drying time is 48-72h.
The oriented nanofibers support of the present invention is made up of the coaxial electrostatic spinning nano fibre loading nerve growth factor (NGF),
Nerve growth factor (NGF) is positioned at the sandwich layer of nanofiber, and conductive materials is positioned at the cortex of nanofiber, and nanofiber presents
" core-shell " structure, nerve growth factor (NGF) slowly discharges during CO2 laser weld, and orientation texture is that nerve cell increases
Growing and provide good microenvironment, additional electro photoluminescence can promote the extension of neural axon, the synergy of three thus accelerans
Repair speed；The Nerve Scaffold that this invention preparation facilities is simple, control parameter less, obtained has good cell compatibility,
It is expected to be applied in next step clinic.
The nano fiber scaffold that the present invention prepares is from additional electro photoluminescence (conducting polymer), biochemistry (nerve growth factor)
And the approach such as the topological structure (orientation guide) needed for nerve regneration promotes CO2 laser weld speed, study additional electro photoluminescence, god
Through the sustained release of growth, and the synergy that orientation texture three is to nerve regneration.
Beneficial effect
(1) it is an advantage of the current invention that to utilize coaxial electrostatic spinning technology cleverly, obtain conduction spacetabs type nano-fiber nerve conduit,
Its good biocompatibility, it is possible to guide nerve cell along oriented growth, the extension of accelerans aixs cylinder, promote impaired
Nerve regneration is repaired；
(2) this method is simple to operate, favorable repeatability, high financial profit.
Accompanying drawing explanation
Fig. 1 electrostatic spinning receives device schematic diagram；
Fig. 2 be fibroin albumen and lactic acid-caprol acton copolymer spinning solution in add the color change of the support of institute after conductive material polyaniline
Change；
The laser co-focusing photo that Fig. 3 loads the nanofiber transmission electron microscope photo (A) of NGF and NGF is distributed in nanofiber
(B)；
Fig. 4 loads the stereoscan photograph of the nanofiber of NGF；
Fig. 5 adult rat adrenal tissue is addicted to chromium neuroma cell (PC-12) growing state of aixs cylinder on support: under being not powered on incentive condition,
The laser co-focusing photo (A) of unsupported nerve growth factor and the laser co-focusing photo (B) of load nerve growth factor；
Adding outside and (apply the voltage of 100mV/cm along differently-oriented directivity) under stimulation conditions, the laser of unsupported nerve growth factor is altogether
Focus on photo (C) and the laser co-focusing photo (D) of load nerve growth factor；
Fig. 6 is cyclic voltammetry curve figure and the fit equation of conductivity type support.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and not
For limiting the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the application appended claims limited range equally.
Embodiment 1
Weigh albumen fibroin 0.1g, lactic acid-caprol acton copolymer 0.3g, be dissolved in 5ml hexafluoroisopropanol (HFIP), lactic acid-
Caprolactone copolymer molecular weight is Mw ≈ 300,000, within 5 hours, mixes with the speed magnetic agitation of 200r/min. and obtains total concentration
The solution of 8% (w/v)；Polyaniline (Mn ≈ 1.5 ten thousand) and the camphorsulfonic acid of 60mg it is separately added into after mixing, then with
Speed magnetic agitation 48h of 300r/min. mixes prepared cortex Static Spinning solution, takes the nerve growth factor of 10 μ g/ml
(NGF) ultra-pure water solution 1ml is as sandwich layer Static Spinning solution.Shell electrostatic spinning solution is loaded syringe, micro-injection
The fltting speed of pump controls to be 1ml/h, and sandwich layer electrostatic spinning solution loads another syringe, the propelling speed of micro-injection pump
Degree controls as 0.2ml/h.The syringe needle that concentric shafts sandwich layer solution used by coaxial electrostatic spinning passes through is No. 8 syringe needles, and shell solution leads to
The syringe needle crossed is No. 16 syringe needles, and syringe needle is connected with the high pressure of 12kV.Connect with the cylinder of a diameter of 5cm of the smooth looping of aluminium foil
Receive nanofibers, motor controls drum rotation speed 4000r/min., and the distance between concentric shafts spinning head and cylinder reception device is
Certain thickness oriented nanofibers film is received on aluminium foil after 5cm, 5h.Above-mentioned support puts into closed container after taking off, and uses body
The ethanol of fraction 75% carries out steam and processes 24h, and the final vacuum that is disposed is dried 48h, removes residual solvent.At above-mentioned support
Upper plantation adult rat adrenal tissue is addicted to chromium neuroma cell (PC-12), and applies the electro photoluminescence of 100mV/cm along fiber orientation directions.
Result shows: in the case of not shocking by electricity, and unsupported divides with the middle axon length of cell on the support of load nerve growth factor
It is not 0.04 μm and 13.9 μm；In the case of clicking on, unsupported with the centre of cell on the support of load nerve growth factor
Axon length is respectively 7.5 μm and 19.7 μm.
Embodiment 2
Weigh albumen fibroin 0.125g, lactic acid-caprol acton copolymer 0.375g, be dissolved in 5ml hexafluoroisopropanol (HFIP), breast
Acid-caprolactone copolymer molecular weight is Mw ≈ 300,000, within 5 hours, mixes with the speed magnetic agitation of 200r/min. and always obtains
The solution of concentration 10% (w/v)；Polyaniline (Mn ≈ 1.5 ten thousand) and the camphorsulfonic acid of 80mg it is separately added into after mixing,
Mix prepared cortex Static Spinning solution with speed magnetic agitation 48h of 300r/min. again, take the nerve growth of 10 μ g/ml
The factor (NGF) ultra-pure water solution 1ml is as sandwich layer Static Spinning solution.Shell electrostatic spinning solution is loaded syringe, trace
The fltting speed of syringe pump controls to be 1ml/h, sandwich layer electrostatic spinning solution loads another syringe, pushing away of micro-injection pump
Entering speeds control is 0.2ml/h.The syringe needle that concentric shafts sandwich layer solution used by coaxial electrostatic spinning passes through is No. 8 syringe needles, and shell is molten
The syringe needle that liquid passes through is No. 16 syringe needles, and syringe needle is connected with the high pressure of 13kV.Rolling with a diameter of 5cm of the smooth looping of aluminium foil
Cylinder receives nanofibers, and motor controls drum rotation speed 4000r/min., concentric shafts spinning head and cylinder receive between device away from
From for 6cm, after 5h, on aluminium foil, receive certain thickness oriented nanofibers film.After above-mentioned support takes off, employing is put into airtight
Container, carrying out steam with the ethanol of volume fraction 75% and process 48h, the final vacuum that is disposed is dried 72h, removes residual solvent.
Claims (9)
1. conduct electricity the preparation method of spacetabs type neural tissue engineering support, including:
(1) fibroin albumen and lactic acid-caprol acton copolymer blend that mass ratio is 25-50:75-50 are dissolved in solvent, it is completely dissolved after stirring and obtains the solution that quality percent by volume is 8-10%, it is subsequently adding polyaniline PANi and camphorsulfonic acid that mass ratio is 1:1, stir and evenly mix, obtain cortex electrostatic spinning solution；In its mediopellis electrostatic spinning solution, the quality percent by volume of polyaniline PANi is 1.2-1.6%；
(2) nerve growth factor is dissolved completely in ultra-pure water, obtains sandwich layer electrostatic spinning solution；
(3) cortex electrostatic spinning solution, sandwich layer electrostatic spinning solution are respectively charged in syringe, carry out coaxial electrostatic spinning, then suffocating treatment, vacuum drying, must conduct electricity spacetabs type neural tissue engineering support.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterised in that: in described step (1), the molecular weight of lactic acid-caprol acton copolymer is Mn≈ 300,000, the molecular weight Mn ≈ 1.5 ten thousand of polyaniline.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterized in that: in described step (1), dissolved stirring time is 5-6h, add polyaniline PANi and camphorsulfonic acid, the time of stirring is 24-48h, and stir speed (S.S.) is 200-300r/min.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterised in that: in described step (2) center core layer electrostatic spinning solution, the concentration of nerve growth factor is 10 μ g/ml.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterized in that: coaxial electrostatic spinning technique in described step (3) is particularly as follows: cortex electrostatic spinning solution loads in syringe, and under trace advances the effect of pump, entrance coaxial device is combined at the annular spaces between the inside and outside capillary spinning nozzle of capillary shower nozzle；Sandwich layer electrostatic spinning solution is loaded syringe, and the dynamic lower entrance coaxial device of effect at micro-injection pump is combined the interior capillary spinning head of capillary shower nozzle, opens electrostatic pressure generator, advances pump, and cylinder receives the motor of device, carries out spinning.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterised in that: in described step (3), coaxial electrostatic spinning technological parameter is: spinning nozzle be internal layer be 8 extra floor be the concentric shafts spinning head of No. 16；The fltting speed of syringe pump is respectively sandwich layer 0.2ml/h and shell 1.0ml/h；Receiving, at concentric shafts spinning head and cylinder, the regulation voltage accessed between device is 12-13kV；The distance that concentric shafts spinning head and cylinder receive between device is 5-6cm；It is 4000r/min that Rolling motor is supplied to the rotating speed of cylinder.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterised in that: in described step (3), suffocating treatment is that ethanol carries out suffocating treatment.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 7, it is characterised in that: the percentage by volume of described ethanol is 75%, and the suffocating treatment time is 24-48h.
A kind of preparation method conducting electricity spacetabs type neural tissue engineering support the most according to claim 1, it is characterised in that: in described step (3), the vacuum drying time is 48-72h.
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Effective date of registration: 20200303 Address after: Room f303, Fudong community Biotechnology Industrial Park, Qingchi street, high tech Zone, Weifang City, Shandong Province Patentee after: Nuoymeier (Shandong) Medical Technology Co., Ltd Address before: 201620 No. 2999 Renmin Road North, Songjiang new town, Shanghai, Songjiang District Patentee before: DONGHUA University |