CN110038166A - A kind of film of curling certainly and preparation method thereof applied to neural restoration - Google Patents
A kind of film of curling certainly and preparation method thereof applied to neural restoration Download PDFInfo
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Abstract
The present invention provides a kind of film of curling certainly and preparation method thereof applied to neural restoration, this from curling film be by A, B and C up of three-layer with a thickness of 60~120 μm of film, A layers of main component are that the indoles mountain valley with clumps of trees and bamboo is green and fibroin albumen, B layers of main component include Cyclic dipeptides and calcium alginate, C layers of main component include Cyclic dipeptides and sodium alginate, the film meets water orientation and curls into continuous hollow pipe, A layers inside, C layers outside, under nearly red light irradiation, closed luminal structure is rapidly become.The present invention provides a kind of new method for nerve docking, casing repairing in neurologic defect, realizes that simple for production, convenient at pipe, size is controllable and application is convenient requires, and has broad application prospects.
Description
Technical field
The present invention relates to a kind of film of neural restoration treatment, in particular to one kind can maintain neurotrosis portion for a long time
The advantageous microenvironment of position and the composition for crimping film certainly, the preparation and application thereof for guaranteeing neural restoration process.
Background technique
Now, the global attack of terrorism, natural calamity are increasingly frequent, and according to incompletely statistics, China increases peripheral nerve newly every year
Case about 1,000,000 are damaged, wherein neurologic defect case about 300,000, and in clinical position, caused by long section neurologic defect
Neurotrosis often prognosis mala and postoperative patient functional rehabilitation is poor, brings heavy negative to society, family and sufferers themselves
Load.In existing research, nerve trachea combines repairing nerve damage frequently as a kind of timbering material combination cell or growth factor,
But due to being fixed at Guan Buliang, at pipe size and the disadvantages of practical application surgical procedure requirement is high limits its extensive use.
Therefore, prepare that a kind of simple for production, convenient at pipe, size is controllable and the convenient nerve trachea of application become there is an urgent need to.
Sodium alginate is a kind of natural polysaecharides compound extracted from kelp or seaweed, only due to containing in its structure
Special stereochemical structure, to show special physicochemical property: biocompatibility, degradability and adhesive property.Alginic acid
The research of sodium just comes across 1881 earliest, Britain chemist Stanford have found brown seaweed hydrochlorate can with thickening solution,
Form gel, biodegradable and easy film forming.The research of sodium alginate film is risen in nineteen ninety, Koen Dewettinck
Et al. discovery sodium alginate frothing capacity and film forming ability after, domestic and international researcher has started to the wide of sodium alginate film
General research.Sodium alginate has good filming performance, is currently used good filming material, but due to its mechanical property
Limitation, there is no further applications in biomedical materials field.It is also untapped at present to go out to can apply to neural restoration
Sodium alginate film.
It can not prepare in advance from clinical treatment angle due to the significant difference of nerve damage portion and degree of injury
The nerve trachea for meeting caliber requirement well, the nerve trachea that in addition, there will be can not form closed pipe for the nerve of non-detachment
Chamber, clinic need a kind of film that can quickly curl into the luminal structure that adaptation defect neural restoration requires certainly, realize fast
The neuroprotection of speed and treatment.
Ideal this kind of film should meet simultaneously the following conditions: good biocompatibility, slewing molding in situ, persistence
Good, the physical and chemical parameters such as mechanical strength and elasticity are suitable for, easily quickly form the suitable closing lumen of caliber.
Drug-delivery preparation in the uterine cavity of requirements above can be met simultaneously by still lacking one kind at present.
Summary of the invention
It is an object of the invention to overcome, " the quick repairing and treating that existing preparation cannot meet neurotrosis simultaneously all is wanted
Ask " bottleneck, provide it is a kind of have quickly from the film for adapting to the luminal structure that defect neural restoration requires is curled into, be real
The quick reparation of existing neurotrosis provides optimal conditions.
Therefore, a kind of curling film certainly applied to neural restoration of the application protection, by the thickness of A, B and C up of three-layer
Degree is 60~120 μm of film, and for A layer main component for the indoles mountain valley with clumps of trees and bamboo is green and fibroin albumen, B layers of main component include Cyclic dipeptides and sea
Calcium alginate, C layers of main component include Cyclic dipeptides and sodium alginate, and the film chance water orientation curls into continuous hollow
Pipe, A layers inside, and C layers outside, under nearly red light irradiation, rapidly becomes closed luminal structure.
Above-mentioned Cyclic dipeptides are preferably ring (aspartic acid-proline) dipeptides.
Above-mentioned A layer is to be attached on B layer surface in a manner of silk column arranged in parallel.
Above-mentioned A layer is to be attached on B layer surface in a manner of the silk column arranged in parallel of single layer, and preferred single layer is arranged in parallel
Silk column mode.
The thickness of above-mentioned film is preferably 80-90 μm.
Glycerol, disodium ethylene diamine tetraacetate is further added in above-mentioned two layer component of B and C.
Electrolyte is further added in above-mentioned two layer component of B and C, antioxidant, binder, pH adjusting agent, stem cell, controls
Treat the factor, the one or more combination of therapeutic agent.
The above-mentioned film from curling film is prepared according to following scheme:
(1) by sodium alginate 0.5g, Cyclic dipeptides 0.01g, glycerol 0.2ml and disodium ethylene diamine tetraacetate 0.072g in beaker
Middle mixing is added 24ml distilled water, is sealed beaker with sealed membrane, and standing is allowed to sufficiently be swollen, and then stirring 2h makes it uniformly
It dissolves, is stood at 4 DEG C for 24 hours, remove bubble in solution.
(2) sodium alginate soln 10mL is extracted with syringe, the casting film-forming on the mold for have surrounding edge is placed in 60 DEG C of constant temperature
It is dried in baking oven, is dried to smooth film.
(3) calcium chloride solution for preparing 2%, is poured on the film of (2), is totally submerged the film, crosslinking Treatment 30 seconds, is handed over
Calcium chloride solution is poured out immediately after the completion of connection processing, blots film surface solution with filter paper, is placed in draught cupboard and air-dries, obtain
The film of B layers He C layers is formed.
(4) fibroin albumen containing 4% is prepared, green according to the indoles mountain valley with clumps of trees and bamboo: the ratio of the mass ratio 1: 400 of fibroin albumen is added
The indoles mountain valley with clumps of trees and bamboo is green, is mixed into solution, and the film B layer surface with electrostatic spinning process in (3) carries out parallel spinning, sufficiently after drying,
Obtain meeting water from the film for curling into luminal structure, with a thickness of 60~100 μm.
Electrolyte is further added in above-mentioned film preparation process, antioxidant, binder, pH adjusting agent, stem cell, controls
Treat the factor, the one or more combination of therapeutic agent.
A kind of above-mentioned reparation for being applied to defect nerve as neural shell material from curling film.
Each component is cooperative compensating and necessary relationship in the technical solution of the application protection, is played a role jointly.
Detailed description of the invention
Fig. 1 is a kind of plan view from curling film applied to neural restoration of the invention
Fig. 2 is a kind of luminal structure formed from after curling film curling applied to neural restoration of the invention
Fig. 3 is a kind of curling film three-decker schematic diagram certainly applied to neural restoration of the invention
Specific embodiment
The specific embodiment of the invention is discussed in detail below.It should be noted that technology described in following embodiments is special
The combination of sign or technical characteristic is not construed as isolated, they can be combined with each other to reach superior technique
Effect.
Preparation of 1 fibroin albumen of embodiment from curling film
Preparation method: designing according to table 1, prepares from the experimental group for crimping film according to following scheme.
(1) by sodium alginate 0.5g, Cyclic dipeptides 0.01g, glycerol 0.2ml and disodium ethylene diamine tetraacetate 0.072g in beaker
Middle mixing is added 24ml distilled water, is sealed beaker with sealed membrane, and standing is allowed to sufficiently be swollen, and then stirring 2h makes it uniformly
It dissolves, is stood at 4 DEG C for 24 hours, remove bubble in solution.
(2) sodium alginate soln 10mL is extracted with syringe, the casting film-forming on the mold for have surrounding edge is placed in 60 DEG C of constant temperature
It is dried in baking oven, is dried to smooth film.
(3) calcium chloride solution for preparing 2%, is poured on the film of (2), is totally submerged the film, crosslinking Treatment 30 seconds, is handed over
Calcium chloride solution is poured out immediately after the completion of connection, blots film surface solution with filter paper, is placed in draught cupboard and air-dries, obtain shape
At A layers and B layers of film.
(4) fibroin albumen containing 4% is prepared, green according to the indoles mountain valley with clumps of trees and bamboo: the ratio of the mass ratio 1: 400 of fibroin albumen is added
The indoles mountain valley with clumps of trees and bamboo is green, is mixed into solution, and the film B layer surface with electrostatic spinning process in (3) carries out parallel spinning, sufficiently after drying,
Obtain meeting water from the film for curling into luminal structure, with a thickness of 60~100 μm.
It designs according to table 1, is prepared from the control group for crimping film according to above-mentioned similar approach.
Composition of the table 1 from curling film experimental group and control group
Note: "/": this component vacancy is represented；" () ": it represents this content and is replaced by content in bracket；1.: ring (asparagus fern
Propylhomoserin-proline) dipeptides；2.: ring (proline-leucine) dipeptides；3.: ring (aspartic-glycine) dipeptides；4.: ring (color
Propylhomoserin-tryptophan) dipeptides；5.: ring (Phe-Trp) dipeptides；6.: Gly-Pro dipeptides；7.: arginine-is sweet
Propylhomoserin-aspartic acid tripeptides.
Physical features of the embodiment 2 from curling film
Curling film experimental group and the control group certainly of embodiment 1, physiological saline is sprayed under 785nm Infrared irradiation, is seen
Examine from curling film formed closing lumen at Guan Nengli, be recorded as the pipe time, respectively measure sealed tube elasticity modulus, as a result
As shown in table 2.
Physical features of the table 2 from curling film experimental group and control group
Embodiment 3 is applied to the effect of neural restoration from curling film in body
(1) peripheral nerve defect in rats model
Animal prepares: healthy adult male SD rat, 220~250g of weight, water of freely ingesting, and adaptive feeding 1 week.
Modeling operation: the preoperative 12h of animal is deprived of food but not water.With small after 10% chloraldurate (0.3mL/100g) intraperitoneal anesthesia
Animal shaving machine shaves off the hair on right leg, and animal is fixed on operation tube for laboratory animal with prone position.The right leg of iodophor disinfection
After operative region, it is multiplexed alcohol wipe one time, is parallel to femur direction in Rat Right side hip region with operating scissors and cuts off skin.To skin
After skin is cut off, i.e., a visible solid mobilizable subcutaneous connective tissue sheath, it is covered on sarolemma, opens flesh with blunt nosed scissors
Film is separated with the biceps muscle of thigh that sarolemma is covered with by the anatomical directions of passivity stringer, can expose right leg sciatic nerve.With hemostasis
After pincers retract two sides muscle with the larger visual field of exposure, the careful peripheral nerve of glass minute hand, apart from pyriformis under greater trochanter of femur
It is starting point at leading edge about 0.5cm, cuts the neural row Sciatic of 1cm.Strictly in accordance with nothing in entire surgical procedure
Bacterium operating principle.
(2) from the restoration effect for crimping film
Using good Sciatic model established above, each group, which is cut into size from curling film, before use is
Two sides broken ends of fractured bone nerve is placed in film by the rectangle of 1.0cm x 0.5cm after peripheral nerve defect in rats, and syringe extracts pre-
Film infiltration is allowed to from wrapping two sides broken ends of fractured bone nerve is rolled up by 37 DEG C of physiological saline of heat, using 785nm near infrared light 20s,
With sutures in pipe both ends sewn reinforcement.By 8 weeks, comparative analysis each group made the reparation of defect nerve from curling film
With.
Step northern blot was carried out to modeling rat in the 8th week after surgery.Before test starts, by two soles of rat hindlimb
Ink is all red-dyed, then putting it into the runway of 1m wide 1.5cm one long makes it walk forward.Each pairs of step
It will all be measured and sciatic nerve function index (SFI) is calculated according to following equation.PL is total vola length, and TS is the first foot
Toe is to the length between the 5th toe, and IT is second toe to the length between the 4th toe, and E represents experimental side, and N is represented
Normal side.SFI closer -100% represents function and all loses, otherwise better closer to 0%.
To each group mouse with row heart perfusion after 10% chloraldurate (0.3mL/100g) intraperitoneal anesthesia after Behavior test
And it draws materials.Experimental side, that is, right sciatic nerves are taken out, 4h are fixed with 4% paraformaldehyde, through 20% and 30% sucrose solution gradient
OCT embeds liquid nitrogen flash freezer after being dehydrated each 12h, and -20 DEG C of refrigerators are sliced after saving overnight using freezing microtome, and slice thickness is 5 μ
m.It takes each group rats with bilateral gastrocnemius and measures weight, calculate gastrocnemius index (GMI) and damage side gastrocnemius and do not damage side
The weight ratio of gastrocnemius is to evaluate amyotrophia caused by injury of sciatic nerve.
By the pathological section of the above rat sciatic nerve, by HE dyeing, immunofluorescence method, measurement defect nerve is repaired
Multiple effect；Each group sciatic nerve electron microscope specimen is cut using transmission electron microscope (TEM) observation, compares and is answered through different groups from curling film
With the tissue morphology variation of rear neural myelin.
Based on result above, using double-blind evaluation each group from the application effect for crimping film, respectively from sciatic nerve function
Whether energy index, gastrocnemius atrophy degree (negativity evaluation index), Histological evaluation (promote neural axon growth and blood vessel again
It is raw), neural myelin recovery extent etc. evaluated, provide comprehensive score (full marks 10 divide), score is higher to represent effect more
It is good.Experimental result is as shown in table 3.
Application effect of the table 3 from curling film experimental group and control group
Experimental group: the behaviouristics step marking and calculate resulting sciatic nerve function index the result shows that, each experiment
Group Damage of Rats side is right side sole skin health, toe differential and SFI is about -50%.Gastrocnemius atrophy degree evaluation hair
It is existing, damage side gastrocnemius with do not damage the weight in wet base of side gastrocnemius than many places in 70% or so, have no apparent damage amyotrophic lateral.
Histological observation shows that damaging side sciatic nerve links together, and neural axon growth and revascularization are obviously promoted.
Find that myelin form is complete, and thickness has a degree of reduction from neural myelin recovery extent.In each experimental group, with experimental group 1
Comprehensive score it is best, followed by experimental group 6, followed by experimental group 2,3,7,8.
The application effect of each control group is significantly lower than experimental group.By the behaviouristics step marking and calculate resulting ischium
Nervous function index is it is found that control rats damage side is that right side sole has and festers and toe is in close up to roll up shape in various degree
State, SFI is about -10%.The evaluation discovery of gastrocnemius atrophy degree, damage side gastrocnemius and the weight in wet base ratio for not damaging side gastrocnemius
Many places are in 20% or so, it is seen that significantly damage amyotrophic lateral.Histological observation shows that damaging side sciatic nerve is not connected
Still in damage condition, axon growth disorder is fresh to there is neural blood vessel regeneration less.It is found from neural myelin recovery extent, myelin
Form is imperfect, and quantity and thickness substantially reduce.In each control group, control group 14 scores highest, followed by control group 2,3,
9,10, and control group 4~6,11~13 is without any effect.
By upper result as it can be seen that of the invention has good repairing effect for defect nerve from curling film, from curling
It is complimentary to one another between film structure and its component, it is indispensable, play synergistic effect.
Above-mentioned detailed description is illustrating for the possible embodiments invented, and the embodiment is not to limit this hair
Bright the scope of the patents, it is all without departing from equivalence enforcement or change of the invention, it should all be contained in the scope of the patents of the invention.
In addition, those skilled in the art can also the claims in the present invention scope of disclosure and spirit in do other forms and
Various modifications, addition and replacement in details.Certainly, these spirit is done according to the present invention various modifications, addition and replacements
It, all should be comprising within scope of the present invention Deng variation.
Claims (10)
1. a kind of curling film certainly applied to neural restoration, it is characterised in that: described is by A, B and C tri- from film is crimped
Layer composition with a thickness of 60~120 μm of film, A layer main component are green and fibroin albumen, B layers of main component include for the indoles mountain valley with clumps of trees and bamboo
Cyclic dipeptides and calcium alginate, C layers of main component include Cyclic dipeptides and sodium alginate, and the film meets water and orients the company of curling into
Continuous hollow pipe, A layers inside, and C layers outside, under nearly red light irradiation, rapidly becomes closed luminal structure.
2. the curling film according to claim 1 certainly applied to neural restoration, it is characterised in that: the Cyclic dipeptides are excellent
It is selected as ring (aspartic acid-proline) dipeptides.
3. it is according to claim 1 applied to neural restoration from curling film, it is characterised in that: the A layer be with
Silk column mode arranged in parallel is attached on B layer surface, preferred single layer silk column mode arranged in parallel.
4. it is according to claim 1 applied to neural restoration from curling film, it is characterised in that: the A layer be with
The silk column mode arranged in parallel of single layer is attached on B layer surface.
5. the curling film according to claim 1 certainly applied to neural restoration, it is characterised in that: the thickness of the film
Preferably 80-90 μm of degree.
6. the curling film according to claim 1 certainly applied to neural restoration, it is characterised in that: the B and two layers of C
Glycerol, disodium ethylene diamine tetraacetate are further added in component.
7. the curling film according to claim 1 certainly applied to neural restoration, it is characterised in that: the B and two layers of C
Electrolyte, antioxidant, binder, pH adjusting agent, stem cell, the treatment factor, one kind of therapeutic agent are further added in component
Or several combinations.
8. a kind of curling film preparation method described in claim 1 certainly applied to neural restoration, it is characterised in that: described
It is prepared from the film of curling film according to following scheme:
(1) sodium alginate 0.5g, Cyclic dipeptides 0.01g, glycerol 0.2ml and disodium ethylene diamine tetraacetate 0.072g are mixed in beaker
It closes, 24ml distilled water is added, is sealed beaker with sealed membrane, standing is allowed to sufficiently be swollen, and then stirring 2h makes its uniform dissolution,
It is stood at 4 DEG C for 24 hours, removes bubble in solution.
(2) sodium alginate soln 10mL is extracted with syringe, the casting film-forming on the mold for have surrounding edge is placed in 60 DEG C of constant temperature ovens
Middle drying is dried to smooth film.
(3) calcium chloride solution for preparing 2%, pours on the film of (2), is totally submerged the film, and crosslinking Treatment 30 seconds, at crosslinking
Calcium chloride solution is poured out immediately after the completion of reason, blots film surface solution with filter paper, is placed in draught cupboard and air-dries, obtain shape
At B layers and C layers of film.
(4) fibroin albumen containing 4% is prepared, green according to the indoles mountain valley with clumps of trees and bamboo: indoles is added in the ratio of the mass ratio 1: 400 of fibroin albumen
The mountain valley with clumps of trees and bamboo is green, is mixed into solution, and the film B layer surface with electrostatic spinning process in (3) carries out parallel spinning and obtains sufficiently after drying
Water is met from the film for curling into luminal structure, with a thickness of 60~100 μm.
9. it is according to claim 7 from curling film preparation method, it is characterised in that: in the film preparation process into
Electrolyte, antioxidant, binder, pH adjusting agent, stem cell, the treatment factor, one or more of groups of therapeutic agent is added in one step
It closes.
10. the curling film according to claim 1 certainly applied to neural restoration, it is characterised in that: described crimps certainly
Film is applied to the reparation of defect nerve as a kind of neural shell material.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021036200A1 (en) * | 2019-08-26 | 2021-03-04 | 北京理工大学 | Method for preparing miniature intelligent calcium alginate hydrogel end operator |
| CN115970064A (en) * | 2023-01-17 | 2023-04-18 | 北京大学人民医院 | Elastic self-curling peripheral nerve repair membrane and preparation method and application thereof |
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| US20020051806A1 (en) * | 2000-04-19 | 2002-05-02 | Mallapragada Surya K. | Patterned substrates and methods for nerve regeneration |
| CN101579246A (en) * | 2009-05-31 | 2009-11-18 | 苏州大学 | Artificial silk fibroin nano-fiber nerve repair conduit and preparation method thereof |
| US20130004559A1 (en) * | 2011-06-30 | 2013-01-03 | Collagen Matrix, Inc. | Flat self-curling permeable sheet membrane |
| CN104548345A (en) * | 2015-01-16 | 2015-04-29 | 上海交通大学 | Elastic self-curling coating nerve-implantable connector device and manufacturing method thereof |
| CN105233344A (en) * | 2015-10-22 | 2016-01-13 | 温州医科大学 | Composite repair material for bridging defect nerves and stent made of composite repair material |
| CN108653815A (en) * | 2017-03-31 | 2018-10-16 | 国家纳米科学中心 | Three-dimensional coil structure and its preparation method and application with autonomous adjustment structure function |
| CN109172036A (en) * | 2018-06-22 | 2019-01-11 | 中山大学附属第医院 | A kind of multichannel peripheral nerve conduit and preparation method thereof |
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| WO2021036200A1 (en) * | 2019-08-26 | 2021-03-04 | 北京理工大学 | Method for preparing miniature intelligent calcium alginate hydrogel end operator |
| CN115970064A (en) * | 2023-01-17 | 2023-04-18 | 北京大学人民医院 | Elastic self-curling peripheral nerve repair membrane and preparation method and application thereof |
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