CN107149699A - A kind of neural tissue engineering conductive fiber tubular bracket and preparation method thereof - Google Patents
A kind of neural tissue engineering conductive fiber tubular bracket and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of neural tissue engineering conductive-nano-fibers tubular bracket and preparation method thereof, the support is made up of conductive-nano-fibers pipe and electric conductivity hydrogel packing material；Conductive-nano-fibers pipe is respectively to be composited by inside and outside along the parallel arrangement conductive-nano-fibers film of conduit axial orientation, non-conductive nonwoven arrangement nano fibrous membrane；Packing material is composited by natural macromolecular hydrogel and growth factor-loaded conductive nanotube.Catheter holder material in the present invention can provide multi-functional bionical microenvironment for neural tissue engineering, with preferable biocompatibility, nutrition goods and materials can be provided for CO2 laser weld, the fiber morphology of two-dimensional orientation can promote the differential growth of nerve cell, and the addition of conductive material can further improve the differentiation degree of nerve cell, promote cell propagation, the required condition of nerve regneration is met well.
Description
Technical field
The present invention relates to technical field of biological material, it is related to a kind of CO2 laser weld nerve trachea and preparation method thereof, especially relates to
And a kind of conductive-nano-fibers tubular bracket and it is a kind of be used for manage it is interior as promote nerve regneration novel tissue engineering filling material
Material and preparation method.
Background technology
Because peripheral nerve tissue's damage that the reasons such as severe trauma, tumor resection are caused is very common in clinic.Nerve
Once it is impaired, will make patient can not normal life and work, but the reparation of big section neurologic defect and reconstruction are still clinical
Problem.Nerve-grafting is the big section neurologic defect method of clinically generally acknowledged relatively effective treatment, but autologous nerve limited source,
Allogeneic nerve is difficult to avoid immunological rejection, replaces nerve to be transplanted with skeletal muscle or vein, and repairing effect is again limited.With god
Development through organizational engineering, biological absorbable nerve trachea turns into the method for being most hopeful successfully to repair big section neurologic defect.
The basic function of nerve trachea is to provide a passage for the growth of nerve, in order to provide preferable microenvironment to axon growth,
Domestic and international researcher is preparing with longitudinal arrangement pore structure nerve trachea, the nerve trachea of load bioactive substance, led
The many-sides such as electrical nerve trachea are furtherd investigate, and these tube materials are it is said that can accelerans growth and reduction nerve
The formation of knurl.But from the angle of organizational engineering, except biocompatibility and biodegradability, repaired for nerve regneration
Ideal stent material should have simultaneously：The topology of the exsule length direction of leading axle；Stimulate axon regeneration and nervous function
The bionical electrophysiological characteristics recovered；Improve the bioactive substance of nerve regneration speed and quality；Load is beneficial to CO2 laser weld
Cell etc..Conditions above is met, completely bionical nerve conduit stent is prepared, the effect of CO2 laser weld will be increased substantially.
In current research, various nerve trachea materials and design are constantly pushed out, and such as patent CN102525689B is used
Method of electrostatic spinning is prepared for a kind of oriented nanofibers nerve trachea, and guiding nerve grows along fiber orientation directions；Patent
CN1303946C discloses the preparation method and its particular manufacturing craft of a kind of neural tissue engineering tubular bracket, constructs inside and carries
The chitosan tubular bracket of axial multi-channel structure；Patent CN101579246A discloses a kind of system of double-deck nerve rehabilitating tube
Standby, the fibroin fiber that its ectonexine is arranged by parallel and vertical conduit extension direction respectively is constituted；Patent
CN103230622A also discloses that a kind of multi-layer fiber film the is built, nerve-grafting that degradation speed slows down gradually from inside to outside is led
Pipe；A kind of pipe body support with conductive capability is prepared using high score conductive material in patent CN10105194729A, passes through body
Electrode is implanted into, applies electro photoluminescence, rat ischium repairing effect is good；A kind of perfluor three is disclosed in patent CN103263695A
The nerve trachea preparation method that ethamine emulsion is combined with seed cell, is to inject the perfluoro triethylamine emulsion for being mixed with seed cell
Obtained in many degradable collagen-chitin nerve tracheas of micropore.But it can be nerve fiber work not yet to have a kind of catheter holder material
Journey provides multi-functional bionical microenvironment.Therefore, how optimization design and to prepare and a kind of more comprehensive promote CO2 laser weld and again
Raw tissue engineering bracket, is the prerequisite for ensureing regenerating nerve functional rehabilitation and giving full play to effect.
The content of the invention
The purpose of the present invention is to provide a kind of scaffold matrices for neural tissue engineering, using electroconductive polymer material
Material, prepares and loads neurotrophic factor and the conductive tubular support beneficial to nerve regneration seed cell simultaneously.The support tube wall
With permeability, but obstruct the entrance growth of fibr tissue；The support inner tubal wall has the topology knot in parallel conduits extension direction
Structure, beneficial to neural axon and the oriented growth of seed cell；Conducting gel layer is filled with the tubular bracket, conduit can be prevented
Structural collapse, and it is used as the seed cell and the carrier of growth factor for promoting nerve regneration.The nerve fiber of this Complex Function
Engineering tubular bracket, cell function is carried out with reference to electro photoluminescence（Such as adhesion, propagation, migration and differentiation）Regulation, can effectively facilitate
Repairing of neural injury.
Foregoing invention purpose is achieved through the following technical solutions：
A kind of neural tissue engineering conductive tubular support, it is characterised in that：The support is by conductive fiber pipe and electric conductivity hydrogel
Packing material is constituted；Conductive-nano-fibers pipe is composited by inside and outside two layers of tunica fibrosa, i.e., outer tube layer is arranged for non-conductive nonwoven
Tunica fibrosa, inner tube layer is the parallel arrangement conductive fiber film along conduit axial orientation；Packing material is by natural macromolecular hydrogel
It is composited with growth factor-loaded conductive nanotube.
Described non-conductive fiber film, its material is biodegradable aliphatic polyester, can be polylactide, the friendship of poly- second
One kind in ester, polycaprolactone and their copolymer.
Described conductive fiber film, its material is conductive polypyrrole（PPY）With being total to for biodegradable aliphatic polyester
Mixed compound.
The one kind of described natural macromolecular hydrogel in sodium alginate or Sodium Hyaluronate.
Described conductive nanotube is PPY nanotubes, a diameter of 300-600 nm, thickness of pipe wall 80-130 nm.
The growth factor of described rush nerve regneration can be nerve growth factor（NGF）And basic fibroblast growth
The factor（bFGF）One or both of.
The seed cell of described rush nerve growth can be between schwann cell, myelin cell, NSC and marrow
One kind in mesenchymal stem cells.
Present invention also offers a kind of preparation method of neural tissue engineering conductive fiber tubular bracket, its step is as follows：
（1）By pyrrole（PY）, dopant DBSA（DBSA）After being well mixed using deionized water dissolving, plus
Enter initiator ammonium persulfate（APS）, trigger PY polymerizations at 0 DEG C, after the completion of polymerization, product is cleaned with methanol, water, acetone respectively
Purifying, chloroform is redissolved in after drying（CHCl3）, it is filtered to remove behind insoluble part, filtrate revolving is removed into solvent, obtained
To drying, soluble PPY.
（2）By step（1）The PPY of preparation, by certain weight ratio, chloroform is dissolved in biodegradable aliphatic polyester jointly
With dimethylformamide（DMF）Mixed solution（Volume ratio 1:1）In, use the metal roller rotated at a high speed for reception device, lead to
Cross electrostatic spinning and prepare the parallel-oriented conductive polyester/PPY composite cellulosic membranes of fiber height.
（3）By step（2）Parallel-oriented polyester/PPY the composite cellulosic membranes of the height of preparation, are existed using aqueous dopamine solution
Room temperature progress surface is modified, is fixed on aluminium foil.
（4）Biodegradable aliphatic polyester is dissolved in trifluoroethanol, using step（3）It is fixed with the aluminium of parallel fibers film
Paper tinsel is reception device, and by electrostatic spinning, the non-woven polyester fabric film of fiber random arrangement is directly being accumulated thereon.
（5）By step（4）The double layer fibre film of preparation, using parallel fibers film as internal layer, perpendicular to fiber orientation directions volume
Qu Chengguan, obtains the fiber body of bilayer.
（6）Biodegradable aliphatic polyester is dissolved in trifluoroethanol, uses aluminium foil for reception device, passes through electrostatic spinning
Nonwoven polyester nano fibrous membrane is prepared, this nano fibrous membrane is soaked in PY monomer solutions, catalyst Fe Cl is added3It is molten
Liquid, PY polymerize and is deposited on polyester nano fiber, then soaks the PPY/ polyester composite nano fibers with chloroform, and dissolving is removed
The PPY nanotubes of conduction are obtained after polyester, drying.
（7）By step (6）After the conductive PPY nanotubes of preparation are using aqueous dopamine solution modification, dry, grinding, sterilizing,
It is scattered to be impregnated in the growth factor aqueous solution through filtration sterilization, by the suction-operated of nanotube come growth factor-loaded.
（8）The aqueous solution of sodium alginate or Sodium Hyaluronate is configured, the seed cell suspension of certain density is added thereto,
And step is added simultaneously（7）The growth factor-loaded PPY nanotubes prepared, obtain mixed liquor A, by the mixed liquor A from step
（5）Nanofiber body one end injection of preparation, sodium alginate or Sodium Hyaluronate are acted on the calcium or magnesium ion in culture medium
Afterwards, gel is formed.
The preparation process（1）In, the dopant ratio of optimization is 0.5（Mol ratio DBSA:PPY）, the monomer of optimization with
The mol ratio of catalyst is 7, and the polymerization time optimized at 0 DEG C is 8h.
The preparation process（2）In, the PPY of optimization and the weight ratio of biodegradable aliphatic polyester are 1:10, matched somebody with somebody
The preferred mass concentration expressed in percentage by volume for putting mixed solution is 20%.
The preparation process（2）In, the metal roller rotated at a high speed, linear resonance surface velocity scope preferably is 12.5 m/s-
20 m/s, the spinning time is not less than 10h, obtains the height-oriented parallel fibers film that thickness is not less than 60um.
The preparation process（2）And step（6）The middle biodegradable aliphatic polyester used, can be identical,
Can be different.
The step（6）The PPY/ polyester composite nano fibers of middle preparation, be one kind using polyester fiber as core, using PPY as
The nuclear shell structure nano fiber of shell, after dissolving the polyester fiber for removing core through chloroform, obtains hollow PPY nanotubes.
The step（3）With the step（7）In, polyester/PPY composite fibres and PPY nanotubes are modified used many
Bar amine aqueous solution, its dopamine solution concentration optimized is 2 mg/ml, and solution ph is 8.5, and the modified-reaction time is 24h.
The step（7）In, PPY nanotubes can load different growth factors respectively, and two kinds of growths can be also loaded simultaneously
The factor.
Invention effect
A kind of conductive fiber tubular bracket that the present invention provides for nerve regneration reparation, it is characterized in that having to design
Property and multifunctionality.The fibrous reticular structure that the fiber tubulose cradle wall has, makes support have permeability, beneficial to nutriment
With the diffusion of metabolic waste；The tube wall of fiber tubular bracket is double-decker, the non-woven fibre film of outer layer, fiber bulk density
Height, can obstruct extraneous fibr tissue and enter growth in pipe, influence CO2 laser weld, the electrical conductive parallel tunica fibrosa of internal layer, with parallel
Axial topology structure in conduit extension direction, beneficial to neural axon and the oriented growth of seed cell, meanwhile, inner fiber film
The conductive characteristic being had, under the stimulation of electric field, can more promote propagation and the differentiation of nerve cell, be conducive to neural process
Growth and extension.The double-decker tube wall of fiber tubular bracket, the biodegradable aliphatic polyester that they are applied can be with
Identical or different, thus can according to nerve regneration to support integrity degree retention time the need for, control support
Tube wall ectonexine has the gradient of consistent degradation rate or degradation rate reduction from inside to outside.
The conducting gel layer of the nanotube containing PPY of filling inside the conducting fibre tubular bracket of the present invention, wherein,
The function one of PPY nanotubes is the controlled release carrier as growth factor, and function two is to confer to the more excellent electric conductivity of gel rubber material,
Function three is that have certain enhancing effect to hydrogel；The function of sodium alginate or Sodium Hyaluronate hydrogel is careful as planting
Born of the same parents and the carrier of growth factor-loaded PPY nanotubes, are that neural tissue engineering kind is careful by the sustained release of growth factor
Born of the same parents provide preferably propagation and differentiation microenvironment.In addition, the nanotube containing PPY of filling is led inside conducting fibre tubular bracket
Coagulation glue material, additionally it is possible to play a part of the soft fiber tubular bracket of support, to prevent collapsing for guide-tube structure, obtain
Nerve regneration repairing effect.
Present disclosure is described in detail below in conjunction with embodiment, but the present invention be not limited to it is following this
A little examples, without departing from the idea case in the present invention described above, make according to ordinary skill knowledge and customary means
Various replacements and change, all should be included within the scope of the invention.
Brief description of the drawings：
The nonwoven of Fig. 1 random arrangements gathers（Lactide coglycolide）Copolymer（PLGA）The SEM figures of fiber.
The SEM figures of the PLGA/PPY composite fibres of parallel arrangement height-oriented Fig. 2.
The TEM figures of Fig. 3 PPY nanotubes.
Embodiment：
Embodiment 1
（1）4 g PY and 10 g DBSA are dissolved in 100 ml deionized waters, are placed in ice bath, under mechanical agitation, thereto by
It is added dropwise to the APS aqueous solution [20% of precooling（w/v）] 10 ml, trigger PY polymerizations in PY/DBSA solution under mechanical stirring, instead
Temperature control is answered at 0 DEG C, after 8h add excessive methanol make reaction terminating, black precipitate is collected by centrifugation, respectively with deionized water,
Methanol, acetone washing, until liquid become colorless it is transparent.Black solid product is dissolved in chloroform, insoluble matter, rotation is filtered to remove
Evaporative removal solvent, prepares soluble PPY.
（2）By 2 g PLGA7525, (ten thousand) mol% of lactide content 75, molecular weight 10 is dissolved in 5.5 ml chloroforms, by 0.2 g
Step（1）The PPY of preparation is dissolved in 5.5 ml DMF, and it is 20% that quality concentration of volume percent is obtained after the mixing of both equal proportions
Mixed solution, rotate at high speed（The m/s of linear resonance surface velocity 16.7）Metal roller be reception device, electrostatic spinning prepares parallel
PLGA/PPY composite cellulosic membrane A, wherein PLGA7525 and PPY weight ratio be 10.Spinning parameter is：The cm of distance 20 is received,
The ml/h of flow velocity 0.4, the kV of voltage 15, the rpm of drum rotation speed 1500.
（3）0.6 g dopamine hydrochlorides are dissolved in 300 ml deionized waters, are 8.5 with Tris regulation pH value of water solution,
By step（2）Parallel-oriented polyester/PPY the composite cellulosic membranes of the height of preparation, are immersed in aqueous dopamine solution, shake at room temperature
Swing reaction treatment 24h.Cleaned after vacuum drying, be fixed on aluminium foil with deionized water after taking-up.
（4）In the trifluoroethanol that 2g PLGA7525 are dissolved in 10 ml, the spinning solution that concentration is 20% is obtained, using step
（3）The aluminium foil for being fixed with parallel fibers film A is reception device, and fiber random arrangement is directly being accumulated thereon by electrostatic spinning
Nonwoven PLGA7525 tunica fibrosas, obtain double layer fibre film B.Spinning parameter is：Receive the cm of distance 20, the ml/h of flow velocity 0.6, voltage
18 kV。
（5）By step（4）The double layer fibre film B of preparation, using parallel fibers film as internal layer, perpendicular to fiber orientation directions volume
Qu Chengguan, suture is fixed and the fiber body of bilayer is obtained with ultraviolet sterilization.Tube length 10-60 mm, internal diameter is 0.5-10
Mm, wall thickness is 0.1-0.3 mm.
（6）Aluminium foil is used for reception device, to step（4）In PLGA7525 solution carry out electrostatic spinning, spinning parameter
Same step（4）, obtain nonwoven nanofiber film C.Configuration concentration is 14 mmol L-1PY and paratoluenesulfonic acid sodium salt mixing water
Solution（Mol ratio is 1:1）.The mm size tunica fibrosas C of 100 mm × 70 is soaked in the above-mentioned mixed solutions of 100 ml, at ultrasound
Manage after 30s, 0 DEG C of hatching 1h, the concentration for adding 50 ml precoolings is 38 mmol L-1Iron chloride（FeCl3）The aqueous solution, shakes at 0 DEG C
Swing polymerization 10h.Take the film out and cleaned with deionized water, then soak 48h with chloroform, take out the PPY nanometers for being dried to obtain conduction
Pipe, a diameter of 300-600 nm, thickness of pipe wall 80-130 nm.
（7）By step (6）The conductive PPY nanotubes of preparation use step（3）Middle aqueous dopamine solution method of modifying processing,
It is fully ground after drying and ultraviolet sterilization.0.01 mg NGF are dissolved in 10 ml deionized waters, filtration sterilization, after sterilizing
0.1 g PPY nanotubes it is scattered be impregnated in the rush nerve growth factor aqueous solution, loaded by the suction-operated of nanotube
Growth factor.
（8）Configuration concentration is 6%（w/v）Step is added after the aqueous solution of Sodium Hyaluronate, filtration sterilization（7）What is prepared is negative
The PPY nanotubes of growth factor are carried, calcic, the Hanks balanced salt solutions of magnesium ion is added（Cellgro companies）, it is well mixed
Concentration is reached 5%, obtain mixed liquor A, by the mixed liquor A from step（5）Nanofiber body one end injection of preparation, it is transparent
Matter acid sodium is with after the calcium in balanced salt solution or magnesium ion effect, forming gel, obtaining a kind of load NGF neural tissue engineering
Conductive fiber tubular bracket.
Embodiment 2：
The conductive fiber tubular bracket of filling conductive hydrogel is prepared using method same as Example 1, except tubular bracket
Outer layer biodegradable polyesters non-woven fibre film, PLGA7525 PLLA is slower than using degradation speed（PLLA, molecular weight
Ten thousand) 10 prepare, and thus obtain fiber tube wall has a kind of nerve fiber work of load NGF of certain degraded gradient from inside to outside
Journey conductive fiber tubular bracket.
Step（4）Operate as follows, other operating procedure be the same as Examples 1：
1g PLLA are dissolved in 10 ml trifluoroethanols, the viscous solution that quality concentration of volume percent is 10% is obtained, will be fixed with
Parallel fibers film A aluminium foil is reception device, and the PLLA fibers of fiber random arrangement are directly being accumulated thereon by electrostatic spinning
Film obtains double-deck spinning fibre film C.Spinning parameter is：Receive the cm of distance 20, the ml/h of flow velocity 0.4, the kV of voltage 15.
Embodiment 3：
The conductive fiber tubular bracket of filling conductive hydrogel is prepared using method same as Example 1, except step（7）Middle institute
The growth factor aqueous solution of configuration is the solution simultaneously containing NGF and bFGF, thus obtains a kind of while two kinds of load promotees nerve
The neural tissue engineering conductive fiber tubular bracket of the regenerative growth factor.
Step（7）Operate as follows, other operating procedure be the same as Examples 1：
By step (6）The conductive PPY nanotubes of preparation use step（3）Middle aqueous dopamine solution method of modifying processing, after drying
It is fully ground and ultraviolet sterilization.0.01 mg NGF and 0.01 mg bFGF are dissolved in 10 ml deionized waters simultaneously, crosses and filters out
Bacterium, scattered be impregnated in of the 0.1g PPY nanotubes after sterilizing is promoted in the nerve growth factor aqueous solution, passes through the absorption of nanotube
Act on loading two kinds of growth factors.
Embodiment 4：
The conductive fiber tubular bracket of filling conductive hydrogel is prepared using method same as Example 1, except being filled out inside it
Schwann cell is loaded in the hydrogel material filled, thus obtain it is a kind of and meanwhile load promote nerve regneration seed cell and growth because
The neural tissue engineering conductive fiber tubular bracket of son.
Step（8）Operate as follows, other operating procedure be the same as Examples 1：
From the preferable schwann cell of growth conditions, configuration concentration is 200/ml cell suspension.Configuration concentration is 6% transparent
The aqueous solution of matter acid sodium, step is added after filtration sterilization（7）The growth factor-loaded PPY nanotubes prepared, add cell and hang
Liquid is well mixed to make hyaluronic acid na concn reach 5%, obtains mixed liquid B, by the mixed liquid B from step（5）The Nanowire of preparation
The injection of body one end is tieed up, thus Sodium Hyaluronate obtains a kind of same with after the calcium in culture medium or magnesium ion effect, forming gel
When load promote nerve regneration seed cell and growth factor neural tissue engineering conductive fiber tubular bracket.
Claims (8)
1. a kind of neural tissue engineering conductive tubular support, it is characterised in that：The support is by conductive-nano-fibers pipe and electric conductivity
Hydrogel packing material is constituted；Conductive-nano-fibers pipe is composited by two layers of nano fibrous membrane, i.e., outer tube layer is non-conductive nothing
Arrangement nano fibrous membrane is spun, inner tube layer is the parallel arrangement conductive-nano-fibers film along conduit axial orientation；Packing material is by day
Right macromolecule hydrogel and growth factor-loaded conductive nanotube are composited.
2. the forming method of neural tissue engineering conductive tubular support comprises the following steps：
（1）Using DBSA as dopant, soluble PPY is prepared, is pressed with biodegradable aliphatic polyester
Certain proportion is made into blend solution, and parallel-oriented conductive fiber film is prepared by electrostatic spinning；（2）In step（1）Prepare
On electrical conductive parallel tunica fibrosa, biodegradable aliphatic polyester non-woven fibre film is prepared using electrostatic spinning, with parallel fibers film
For internal layer, film is curled into pipe perpendicular to parallel fibers direction, the tunica fibrosa body of bilayer is obtained；（3）By chemical deposition,
Polyester fiber, is completely dissolved after removing by uniform deposition PPY with organic solvent on biodegradable polyesters fiber, obtains conduction
PPY nanotubes；（4）Configuration promotees the growth factor aqueous solution of nerve regneration, and PPY nanotubes are immersed, and takes out and is obtained after drying
Obtain growth factor-loaded PPY nanotubes；（5）The natural polymer aqueous solution is configured, and adds the kind for promoting nerve regneration wherein
Daughter cell, growth factor-loaded PPY nanotubes, step is injected into by the mixed solution（2）The tunica fibrosa tube cavity of preparation,
After gel, the neural tissue engineering conductive tubular support is produced.
3. biodegradable aliphatic polyester according to claim 1, can be polylactide, PGA, gather in oneself
One kind in ester and their copolymer.
4. the one kind of natural macromolecular according to claim 1 in sodium alginate or Sodium Hyaluronate.
5. conductive nanotube according to claim 1 is polypyrrole nanotube, a diameter of 300-600nm, thickness of pipe wall 80-
130 nm。
6. the growth factor according to claim 1 for promoting nerve regneration, can be nerve growth factor（NGF）With alkalescence into
Fibroblast growth factor（bFGF）One or both of.
7. the seed cell according to claim 1 for promoting nerve regneration, can be schwann cell, myelin cell, nerve cord
One kind in cell and mesenchymal stem cells MSCs.
8. in preparation method according to claim 2, it is characterised in that step（1）With（2）Employed in biology can drop
It can be identical or different to solve aliphatic polyester.
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