CN103083724A - Preparation method of nervous tissue repair scaffold loaded with dual trophic factors including ganglioside (GM1) and nerve growth factor (NGF) - Google Patents
Preparation method of nervous tissue repair scaffold loaded with dual trophic factors including ganglioside (GM1) and nerve growth factor (NGF) Download PDFInfo
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- CN103083724A CN103083724A CN2013100395196A CN201310039519A CN103083724A CN 103083724 A CN103083724 A CN 103083724A CN 2013100395196 A CN2013100395196 A CN 2013100395196A CN 201310039519 A CN201310039519 A CN 201310039519A CN 103083724 A CN103083724 A CN 103083724A
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Abstract
The invention relates to a preparation method of a nervous tissue repair scaffold loaded with dual trophic factors including ganglioside (GM1) and nerve growth factor (NGF). The method comprises the processes of constructing a preparation machine, preparing GM1 and NGF ultrapure water, preparing a hexafluoroisopropanol (HFIP) mixed solution of silk fibroin and polylactic acid-polycaprolactone (P(LLA-CL)), preparing the nanofiber scaffold loaded with the dual trophic factors including the GM1 and the NGF, and the like. The prepared nanofiber scaffold reforms a neural repair conduit through a biochemical method (namely addition of the active trophic factors) and a topological structure method (namely introduction of an oriented structure), aims to research the controlled-release behaviors and the nerve regeneration promotion function of the dual trophic factors, and can induce nerve regeneration through nanofiber orientation. The method provides experimental and theoretical basis for neural repair, and provides a new method for clinical repair of large nerve defects.
Description
Technical field
The invention belongs to neurologic defect reparation biomaterial scaffolds and preparation field thereof, particularly the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 and NGF.
Background technology
Peripheral nerve injury is clinical common diseases, hinder from disconnected for perineural clinically, the normal microsurgical technique that adopts is held-holds adventitia stitching, perineurial suture or unites stitching, to recover neural seriality, for perineural damaged, nerve autograft is still " golden standard " of present reparation.In recent years, the rise of organizational project is for numerous tissue defects, patient's organ failure treatment has brought dawn.
The nerve regeneration conduit that utilizes biomaterial to build can obtain nutrition for neurocyte, metabolism and growth provides a favourable space.Yet neuranagenesis is the interactional result of cell, extracellular matrix and neurotrophic factor, single nerve trachea is limited to the repairing effect of injured nerve, because peripheral nerve regeneration not only will recover its structure, the more important thing is and recover its sensation and motor function, in order to meet or exceed the repairing effect of nerve autograft, Recent study person constantly endeavour the preparation of controllable active composite nerve conduit.Report nerve growth factor (Nerve growth factor is arranged in recent years, NGF) can play a driving role to motorial regeneration, this research group has also been reported with coaxial co spun technology in 2008 nerve growth factor has been embedded in nanofiber, makes it slowly to discharge to obtain active nerve trachea.But independent NGF is still fainter to the effect of motor neuron, and the commercially available price of NGF is higher.Therefore, introduce the another kind of trophic factors that NGF is had collaborative mediation, develop the lower and active nerve trachea that have a controlled release model of a kind of cost and have multi-meaning.To so far, there are no preparing Ganglioside GM1, nerve growth factor, the two trophic factors loaded with nano fibers of NGF based on coaxial electrostatic spinning technology, and finally prepare the report of the active nerve rehabilitating tube of the two trophic factors loads of GM1, NGF.
Summary of the invention
technical problem to be solved by this invention is to provide a kind of GM1, active nerve rehabilitating tube of the two trophic factors loads of NGF and preparation method thereof, nano fiber scaffold is by load GM1, the two trophic factors of NGF and the coaxial electrostatic spinning nano fibre with orientation texture consist of, GM1, present " core-shell " structure in the nanofiber of the two trophic factors loads of NGF, two kinds of trophic factors slowly discharge during nerve recovery, induce and accelerate the regeneration of injured nerve, and the nanofiber with orientation texture provides correct direction for neuranagenesis, make the neural axon can oriented growth, accelerated nerve recovery speed, this invention preparation facilities is simple and easy, the control parameter is less, easy and simple to handle, preparation process stable, is expected to be widely used in the preparation process of nerve rehabilitating tube.
The preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 of the present invention and NGF comprises:
(1) GM1 and two kinds of trophic factors of NGF are dissolved in ultra-pure water altogether with mass ratio 1:1, dissolving obtains the solution of every milliliter of 10-20 microgram fully;
(2) be that fibroin albumen and the polylactic acid-polycaprolactone blend of 25:75 is dissolved in hexafluoroisopropanol with mass ratio, obtain the solution that the quality percent by volume is 8-10% after dissolving stirs fully;
(3) solution that step (1) the is obtained propelling container of packing into enters the interior capillary spinning nozzle of the compound capillary shower nozzle of coaxial device under the driving of micro-injection pump; The solution that step (2) the is obtained propelling container of packing into is at the annular spaces place that enters under the driving of micro-injection pump between the inside and outside capillary spinning nozzle of the compound capillary shower nozzle of coaxial device;
(4) start boost pump, voltage and motor, begin to form the coaxial electrostatic spinning nano fibre that orientation receives, obtain described support;
(5) above-mentioned support is taken off rear employing one hermetic container steam treatment, the final vacuum drying that is disposed is removed residual solvent.
Preserve under 4 ℃ of conditions after the ultra-pure water solution preparation of the middle GM1 of described step (1) and NGF is complete, avoid the forfeiture of trophic factors activity.
Polylactic acid in described step (2)-polycaprolactone molecular weight is Mw ≈ 300,000.
The stirring means that adopts in described step (2) is magnetic agitation, and mixing speed is 400 ~ 600 rpms, and mixing time is 12-24 hour.
Spinning nozzle in described step (4) is the composite capillary of concentric shafts, and prepared nanofiber has " core-shell " structure.
Spinning parameter related in described step (4) is: spinning nozzle is that internal layer is that 8 extra floor are the concentric shafts spinning head of No. 16; The fltting speed of syringe pump be respectively 0.2 milliliter of sandwich layer per hour with 1.0 milliliters of shells per hour; The regulation voltage that accesses between concentric shafts spinning head and rotary drum accepter is 12 kilovolts; Distance between concentric shafts spinning head and rotary drum accepter is 15-20 centimetre; The rotating speed that motor offers rotary drum is 4000 rpms.
Steam treatment in described step (5) adopts the ethanol of volume fraction 75%; Processing time is 24-36h.
Be 72-96h the drying time in described step (5).
Can be used as large animal length apart from a kind of GM1 mediation NGF of neurologic defect reparation and have the support of trophic factors controlled release model.
The present invention is based on coaxial electrostatic spinning technology, as cortical material, the surface hydrophilicity that had both improved fiber makes again it have certain mechanical property so that the cell recognition site to be provided with the blend of fibroin albumen and P (LLA-CL); With contain have synergistic pair of trophic factors (GM1 and NGF) aqueous solution as core material, the active nano fiber of preparation skin-core structure, the slow release behavior of the two trophic factors of research reaches the facilitation to neuranagenesis, and induces neuranagenesis by the orientation of nanofiber.
Beneficial effect
(1) neural recovery support of the present invention will have synergistic pair of trophic factors (GM1 and NGF) bag and carry nanofiber, utilize the cylinder of high speed rotating to receive the acquisition orientation texture in preparation process, thereby from biochemical function and topological structure two aspect research neuranagenesis mechanism, for the nerve reparation provides experiment and theoretical foundation.And the active nerve trachea of preparing can be used for repairing large section neurologic defect, discloses the slow release of trophic factors and the mechanism that the fibre orientation inducing action is repaired longer neurologic defect;
(2) preparation facilities involved in the present invention is simple and easy, procedure parameter is few, preparation process is stable, has a wide range of applications in the preparation field of the neural recovery support of activity.
Description of drawings
Nervous tissue's recovery support of the two trophic factors of Fig. 1 load GM1 and NGF prepares schematic diagram;
The scanning microscope photo of the orientation nervous tissue recovery support of the two trophic factors of Fig. 2 load GM1 and NGF;
The nanofiber transmission electron microscope photo of the two trophic factors of Fig. 3 load GM1 and NGF.
The specific embodiment
In conjunction with specific embodiments, further set forth the present invention.Should be understood that these embodiment only are used for explanation the present invention, be not used in to limit the scope of the invention.Should be understood that in addition present technique personnel can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the appended claims limited range of the application equally.
Embodiment 1
Take this albumen fibroin 0.20 gram, polylactic acid-polycaprolactone 0.60 gram is dissolved in 10 milliliters of hexafluoroisopropanols (HFIP), and polylactic acid-polycaprolactone molecular weight is M
w≈ 300,000, obtain the shell electrostatic spinning solution of total concentration 8% with 12 hours mix homogeneously of the speed magnetic agitation of 400 rpms; Get 1 milliliter respectively of nerve growth factor (NGF) the ultra-pure water solution of every milliliter of ganglioside (GM1) the ultra-pure water solution of every milliliter of 10 microgram and 10 microgram, mix homogeneously obtains two milliliters of GM1, the NGF concentration ratio is the 1:1 mixed solution, obtains sandwich layer Static Spinning solution.With the shell electrostatic spinning solution syringe of packing into, the fltting speed of micro-injection pump per hour is controlled to be 1 milliliter, and with sandwich layer electrostatic spinning solution another syringe of packing into, the fltting speed of micro-injection pump per hour is controlled to be 0.2 milliliter.The syringe needle that coaxial electrostatic spinning concentric shafts sandwich layer solution used passes through is No. 8 syringe needles, and the syringe needle that shell solution passes through is No. 16 syringe needles, and syringe needle is connected with the high pressure of 12 kilovolts.Be that the cylinder of 5 centimetres receives nanofibers with the diameter of the smooth looping of aluminium foil, cylinder is connected with motor, 4000 rpms of Electric Machine Control drum rotation speeds, distance between concentric shafts spinning head and rotary drum accepter is 15 centimetres, receives certain thickness oriented nanofibers film after 10 hours on aluminium foil.The ethanol that above-mentioned support takes off rear employing one hermetic container, volume fraction 75% carries out steam treatment 24h, and the dry 72h of the final vacuum that is disposed removes residual solvent.
Embodiment 2
Take albumen fibroin 0.25 gram, polylactic acid-polycaprolactone 0.75 gram is dissolved in 10 milliliters of hexafluoroisopropanols (HFIP), and polylactic acid-polycaprolactone molecular weight is M
w≈ 300,000, obtain the shell electrostatic spinning solution of total concentration 10% with 12 hours mix homogeneously of the speed magnetic agitation of 600 rpms; Get 1 milliliter respectively of nerve growth factor (NGF) the ultra-pure water solution of every milliliter of ganglioside (GM1) the ultra-pure water solution of every milliliter of 20 microgram and 20 microgram, mix homogeneously obtains two milliliters of GM1, the NGF concentration ratio is the 1:1 mixed solution, obtains sandwich layer Static Spinning solution.With the shell electrostatic spinning solution syringe of packing into, the fltting speed of micro-injection pump per hour is controlled to be 1 milliliter, and with sandwich layer electrostatic spinning solution another syringe of packing into, the fltting speed of micro-injection pump per hour is controlled to be 0.2 milliliter.The syringe needle that coaxial electrostatic spinning concentric shafts sandwich layer solution used passes through is No. 8 syringe needles, and the syringe needle that shell solution passes through is No. 16 syringe needles, and syringe needle is connected with the high pressure of 12 kilovolts.Be that the cylinder of 5 centimetres receives nanofibers with the diameter of the smooth looping of aluminium foil, cylinder is connected with motor, 4000 rpms of Electric Machine Control drum rotation speeds, distance between concentric shafts spinning head and rotary drum accepter is 20 centimetres, receives certain thickness oriented nanofibers film after 10 hours on aluminium foil.The ethanol that above-mentioned support takes off rear employing one hermetic container, volume fraction 75% carries out steam treatment 36h, and the dry 96h of the final vacuum that is disposed removes residual solvent.
Claims (8)
1. the preparation method of nervous tissue's recovery support of the two trophic factors of a load GM1 and NGF comprises:
(1) GM1 and two kinds of trophic factors of NGF are dissolved in ultra-pure water altogether with mass ratio 1:1, dissolving obtains the solution of every milliliter of 10-20 microgram fully;
(2) be that fibroin albumen and the polylactic acid-polycaprolactone blend of 25:75 is dissolved in hexafluoroisopropanol with mass ratio, obtain the solution that the quality percent by volume is 8-10% after dissolving stirs fully;
(3) solution that step (1) the is obtained propelling container of packing into enters the interior capillary shower nozzle of the compound capillary shower nozzle of coaxial device under the driving of micro-injection pump; The solution that step (2) the is obtained propelling container of packing into is at the annular spaces place that enters under the driving of micro-injection pump between the inside and outside capillary spinning nozzle of the compound capillary shower nozzle of coaxial device;
(4) start boost pump, voltage and motor, begin to form the coaxial electrostatic spinning nano fibre that orientation receives, obtain support;
(5) above-mentioned support is taken off rear employing one hermetic container steam treatment, the final vacuum drying that is disposed is removed residual solvent.
2. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, it is characterized in that: preserve under 4 ℃ of conditions after the ultra-pure water solution preparation of the middle GM1 of described step (1) and NGF is complete, avoid the forfeiture of trophic factors activity.
3. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, it is characterized in that: the polylactic acid in described step (2)-polycaprolactone molecular weight is M
w≈ 300,000.
4. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, it is characterized in that: the stirring means that adopts in described step (2) is magnetic agitation, mixing speed is 400 ~ 600 rpms, and mixing time is 12-24 hour.
5. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, it is characterized in that: the spinning nozzle in described step (4) is the composite capillary of concentric shafts, and prepared nanofiber has " core-shell " structure.
6. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, it is characterized in that: spinning parameter related in described step (4) is: spinning nozzle is that internal layer is that 8 extra floor are the concentric shafts spinning head of No. 16; The fltting speed of syringe pump be respectively 0.2 milliliter of sandwich layer per hour with 1.0 milliliters of shells per hour; The regulation voltage that accesses between concentric shafts spinning head and rotary drum accepter is 12 kilovolts; Distance between concentric shafts spinning head and rotary drum accepter is 15-20 centimetre; The rotating speed that motor offers rotary drum is 4000 rpms.
7. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, is characterized in that: the ethanol of the steam treatment employing volume fraction 75% in described step (5); Processing time is 24-36h.
8. the preparation method of nervous tissue's recovery support of the two trophic factors of a kind of load GM1 according to claim 1 and NGF, it is characterized in that: be 72-96h the drying time in described step (5).
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Cited By (12)
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| CN104225685A (en) * | 2014-09-18 | 2014-12-24 | 东华大学 | Preparation method for conduction and sustained release type nervous tissue engineering scaffold |
| CN104963027A (en) * | 2015-06-06 | 2015-10-07 | 李松群 | Coaxial electrospinning method of fibroin-polycaprolactone bi-component ultrafine fiber |
| CN104998297A (en) * | 2014-04-16 | 2015-10-28 | 烟台隽秀生物科技有限公司 | Poly(L-lactide-co-epsilon-caprolactone)nano-fiber nerve conduit and preparation method thereof |
| CN106390196A (en) * | 2016-09-07 | 2017-02-15 | 东华大学 | Preparation method of nanofiber nerve tissue engineering scaffold |
| CN106400312A (en) * | 2016-09-07 | 2017-02-15 | 东华大学 | Method for preparing conductive composite nanofiber nervous tissue engineering scaffold based on graphene |
| CN106400311A (en) * | 2016-09-07 | 2017-02-15 | 东华大学 | Method for preparing composite nanofiber tissue engineering scaffold based on graphene oxide |
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| CN106400311A (en) * | 2016-09-07 | 2017-02-15 | 东华大学 | Method for preparing composite nanofiber tissue engineering scaffold based on graphene oxide |
| CN106400312A (en) * | 2016-09-07 | 2017-02-15 | 东华大学 | Method for preparing conductive composite nanofiber nervous tissue engineering scaffold based on graphene |
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| CN114767928A (en) * | 2021-01-22 | 2022-07-22 | 北京化工大学 | Preparation method of nerve conduit loaded with active particles with uniform concentration and nerve conduit |
| CN113509593A (en) * | 2021-05-08 | 2021-10-19 | 武汉理工大学 | Preparation method of directional nanofiber nerve conduit |
| CN113171495A (en) * | 2021-05-31 | 2021-07-27 | 马腾 | Preparation method of gravity-drawn encapsulated microtubule nerve repair scaffold |
| CN113171495B (en) * | 2021-05-31 | 2022-05-03 | 马腾 | Preparation method of gravity-drawn encapsulated microtubule nerve repair scaffold |
| CN113648463A (en) * | 2021-07-22 | 2021-11-16 | 上海市第六人民医院 | Fullerol loaded polycaprolactone nerve scaffold and preparation method thereof |
| CN113975460A (en) * | 2021-11-04 | 2022-01-28 | 中国人民解放军国防科技大学 | Bone repair scaffold material capable of mediating neurogenesis and preparation method and application thereof |
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Application publication date: 20130508 |