CN110218344A - A kind of elastic hydrogel fiber and its preparation method and application - Google Patents
A kind of elastic hydrogel fiber and its preparation method and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
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- A61L2400/00—Materials characterised by their function or physical properties
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- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The present invention provides a kind of elastic hydrogel fiber and its preparation method and application, elastic hydrogel fiber provided by the invention, by obtaining acrylic modified gelatin by electrostatic spinning；It is found through experiments that, the aquagel fibre not only good biocompatibility of obtained modified gelatin preparation, it is soft, and resistance change ability is good, and the high-moisture percentage characteristic with similar cell epimatrix and the performance with micro-nano directional fiber, so that the material be made to have outstanding myeloid tissue's repair function.
Description
Technical field
The present invention relates to technical field of biological material more particularly to a kind of elastic hydrogel fiber and its preparation method and application.
Background technique
The bionical biomaterial facing challenges always of the structure and performance of biological tissue, the regeneration with bionical characteristic
Timbering material is more advantageous to injury tissue reconstruction and functional rehabilitation.Wherein, High water cut, softness, it is porous and have orientation microcosmic three
The mechanics of biological tissue of dimension structure is the challenge of bionical regeneration bracket.For example, myeloid tissue is surrounded by cerebrospinal fluid, and protected
Protect in the canalis spinalis of bony structure, property is soft, by the orientation nerve fibre bundle of uplink and downlink therein reach brain with
Signal connection between different segment spinal cord.Therefore the physiology anatomical features based on spinal cord, biological support needs to have following
Condition: needed for good biocompatibility promotes neural cell adhesion, High water cut and high-permeability to meet cell metabolism,
The three-dimensional structure (3D) of porous orientation is convenient for cell migration and guides axon growth.It is similar with natural extracellular matrix
The mechanical characteristics of flexibility and elasticity provide to maintain the 3D microstructure characteristic of bracket for cell growth and axon elongation
Space.Currently, the regeneration bracket of spinal cord reparation primarily focuses on a certain item therein, for example, there is researcher to use orientation
Very low power or microtube structure imitate spinal cord physiology oriented structure to reach, and guide neural axon growth.Some uses water-setting
Glue, which obtains high-moisture percentage, makes nerve cell obtain sufficient nutrient.But due to the similar such mechanics of biological tissue of spinal cord and property
The complexity of energy, is difficult to realize the abundant bionical of the structure of regeneration bracket up to now.
Organizational project repairs the myeloid tissue of local damage, using exogenous timbering material, in conjunction with or do not combine various
Functioning cell is transplanted in damage location, achievees the purpose that reparation.This artificial material for just needing to transplant imitates naturally to greatest extent
Extracellular matrix, and the characteristic of the mechanical characteristic of the tissue softness based on spinal cord and orientation texture, building good biocompatibility, tool
There are three-dimensional orientation structure, softness and the biological support with certain elasticity, so that promoting spinal cord reparation is regenerative medicine stent cover
The challenge faced.
Hydrogel is applied to group due to having the High water cut and variable mechanical characteristics of similar soft tissue, by more and more
In weaver's journey.The regulation of its water content may be implemented by adjusting its concentration, the degree of cross linking, gel time etc..However, due to water-setting
The bulk characteristic of glue is difficult to realize on the directional fibre structure of especially bionical spinal cord in orientation texture.Electrostatic spinning technique
Be using high voltage power supply by synthetic material liquid pull into micro nanometer fiber as extracellular matrix fiber-like, be further formed
Biologic bracket material can promote cell adherence, migration.Directional fiber can be prepared using the technology, realizes that bionical spinal cord is fine
Tie up the micro nanometer fiber binding structure of structure.But the material of electrostatic spinning fiber mainly passes through the dissolution of high molecular material, melts at present
Melt the building to realize micro nanometer fiber, such material is directly solidified into fibre structure after spinning moulding, but synthesized polymer
Although object material meets different requirements in support Design, there is no cell recognition binding sites for the fiber, to limit
Its application is made.In addition, the electrospinning of water soluble polymer is poor and shaping fiber easy to absorb moisture and redissolution, it is difficult to tie up
Hold its fibre structure and performance, it is difficult to realize combining for fiber and hydrogel, it more difficult to realize the mechanics such as its soft and high resiliency
Performance.
Due to the complexity of similar spinal cord such mechanics of biological tissue and performance, filling for the structure of regeneration bracket is realized
It is point bionical, so that promoting spinal cord reparation is regenerative medicine bracket facing challenges.
Summary of the invention
In view of this, technical problem to be solved by the present invention lies in provide a kind of elastic hydrogel fiber and its preparation side
Method and application, present invention offer elastic hydrogel fiber biological compatibility is good, aqueous abundant and soft with elasticity, can be used as life
Object timbering material is used for the reparation of spinal cord.
Compared with prior art, the present invention provides a kind of elastic hydrogel fiber and its preparation method and application, this hairs
The elastic hydrogel fiber of bright offer, by obtaining acrylic modified gelatin by electrostatic spinning；It is sent out by experiment
Existing, the aquagel fibre of obtained modified gelatin preparation not only good biocompatibility is soft, and to become ability good for resistance, and has
The high-moisture percentage characteristic of similar cell epimatrix and performance with micro-nano directional fiber, so that the material be made to have remarkably
Myeloid tissue's repair function.
Detailed description of the invention
The preparation process and electrospinning that Fig. 1 is acrylic modified gelatin GelMA synthesize elastic hydrogel fiber and are used in combination
In the procedure chart of cell experiment and zoopery；
Fig. 2 is the water of orientation GelMA aquagel fibre provided by the invention and the modified Gelatin of unused methylbenzene olefin(e) acid
The SEM of gelatinous fibre schemes；
Fig. 3 is GelMA and orients the drying test result of gelatin aquagel fibre；
Fig. 4 is GelMA and orients the external degradation test result of gelatin aquagel fibre；
Fig. 5 is GelMA and orients the mechanical experimental results of gelatin aquagel fibre；
Fig. 6 is cell dead test living and CCK-8 cell Proliferation vigor test result；
Fig. 7 is the fluorogram that cell is grown on tunica fibrosa；
The cell migration that Fig. 8 Laser Scanning Confocal Microscope is shown enters internal stent depth map；
Fig. 9 is that GelMA elastic hydrogel fibrous framework repairs result figure to mouse spinal cord；
Figure 10 is GFAP the and CSPG immunofluorescence dyeing that marker glial scar is formed；
Figure 11 is to indicate the CD31 immunofluorescence dyeing of vascular endothelial cell formation and count histogram and evaluate animal
The BBB of postoperative lower limb function recovery situation scores.
Specific embodiment
The present invention provides a kind of elastic hydrogel fibers, are obtained by acrylic modified gelatin by electrostatic spinning
It arrives.
According to the present invention, elastic hydrogel fiber of the present invention passes through electrostatic spinning by acrylic modified gelatin
It obtains；Wherein, the grafting rate of methacrylic acid is 80~100%, more preferably 90 in the acrylic modified gelatin
~100%.
In the present invention, electrostatic spinning of the present invention is preferred specifically:
1) acrylic modified gelatin solution is placed on syringe pump, carries out electrospinning, obtains tunica fibrosa or fibre bundle；
Wherein, the solvent in the solution of the acrylic modified gelatin is preferably hexafluoroisopropanol；The methacrylic acid changes
Property gelatin solution in the concentration of the modified gelatin of olefin(e) acid be preferably 8~12wt%, more preferably 10wt%；The tool of the electrospinning
Body parameter are as follows: 10~20kv high-voltage electricity, preferably 15~18kv high-voltage electricity are flat placing at 18~22cm of syringe nozzle
Row electrode places parallel pole, parallel pole 2~4cm of spacing, preferably in 3cm, with 0.05 after powering on preferably at 20cm
The injection of~0.15ml/min speed, preferably jet velocity are 0.1ml/min；The fibre bundle is the circle crimped by tunica fibrosa
Columnar fiber beam.
2) obtained tunica fibrosa or fibre bundle are crosslinked, obtain elastic hydrogel fiber；Wherein, the crosslinking is preferred
For ultraviolet light cross-linking；Specifically, tunica fibrosa or fibre bundle are preferably placed in photo-crosslinking liquid by the present invention, by ultraviolet light,
Obtain elastic hydrogel fiber；Wherein, the photo-crosslinking liquid is made of photoinitiator and solvent, and the photoinitiator is preferably 2-
Hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone solution (photoinitiator 2959)；The solvent is preferably ethyl alcohol；The light
The concentration of photoinitiator is 3~8wt%, more preferably 5~6wt% in crosslinked fluid；The ultraviolet light intensity is 8~15mW/
cm2, more preferably 10mW/cm2, the reaction time is 50~100min, preferably 60~80min.
In the present invention, preparation method obtains the acrylic modified gelatin in accordance with the following methods:
Methacrylic acid anhydride reactant is added dropwise into gelatin solution, obtains reaction solution；
Reaction solution is dialysed, filters, obtains acrylic modified gelatin,
Wherein, the molecular cut off of the dialysis bag filter is 12~14kDa, and filtering is 0.22um filter membrane with filter membrane.
The present invention also provides a kind of preparation methods of elastic hydrogel fiber, by the modified gelatin of methacrylic acid olefin(e) acid
Elastic hydrogel fiber is obtained by electrostatic spinning；Wherein, each technological parameter of preparation is as hereinbefore.
A kind of tissue the present invention also provides elastic hydrogel fiber of the present invention in preparation for spinal cord reparation
Application in regeneration support.
The present invention provides a kind of elastic hydrogel fiber and its preparation method and application, elasticity water-setting provided by the invention
Glue fiber, by obtaining acrylic modified gelatin by electrostatic spinning；Wherein, by making olefin(e) acid modified gelatin, and
Elastic hydrogel fiber is further prepared, is found by detection, which not only remains a large amount of bio-active groups, such as
Promote arginine-glycine-aspartic acid (RGD) sequence of cell adherence and the matrix of cell remodeling
Metalloproteinase (MMP) sequence, the i.e. modification of methacrylic acid do not have an impact these functional groups.For nerve
For stem cell, the extracellular matrix of low elastic modulus is conducive to Neural differentiation, and in the extracellular of relative rigid
It can be more likely to break up to spongiocyte in matrix.In addition to this, GelMA (Gel indicates glue, MA methacrylic acid group,
GelMA is the abbreviation of methacrylated gelatin) hydrogel due to obtaining better elastic property, can to anti-cell with
And the mechanics influence that surrounding tissue applies it repeatedly.I.e. the present invention passes through the gelatin modified GelMA water-setting for synthesizing Photocrosslinkable
Glue material, and be dissolved in organic solvent construct can electrostatic spinning solution.Then, GelMA electrostatic spinning fiber is being handed over
Photo-crosslinking is carried out in connection agent, then impregnates and constructs hydrogel electrostatic spinning fiber bracket in aqueous solution.In vitro experiment, by right
The micromorphology of bracket, moisture content, mechanical performance, degradation property are studied, at the same to the biocompatibility of timbering material,
Bracket studies the guided bone and promotion cell transfer ability in 3D bracket in cellular prion protein direction.In order into
One step confirms that the orientation resilient three-dimensional nano-bracket material to the repairing effect of spinal cord, is also implanted into rat spinal cord hemisection model
In, histologic analysis and motor function analysis are carried out, as a result, it has been found that, the aquagel fibre of obtained modified gelatin preparation is not only given birth to
Object compatibility is good, soft, and to become ability good for resistance, and the high-moisture percentage characteristic with similar cell epimatrix and with micro-nano
The performance of directional fiber, so that the material be made to have outstanding myeloid tissue's repair function.
It is clearly and completely described below in conjunction with the technical solution of the embodiment of the present invention, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
(1) synthesis of GelMA (acrylic modified gelatin)
20g gelatin is weighed, be added in 2L conical flask and 200mL PBS is added.Conical flask is placed in 60 DEG C of water-baths, is stirred
It mixes to gelatin and is completely dissolved.After gelatin is completely dissolved, methacrylic anhydride is added dropwise into conical flask, amounts to 16mL, it is whole
A dropwise addition process continues 1h.After completion of dropwise addition 2h, the 800mL PBS for being preheated to 50 DEG C is added in conical flask, then is persistently stirred
Mix 15min.The liquid in conical flask is poured into bag filter (Mw:8000-14000) after 15min, dialysis procedure continues one week.
After a week, collect bag filter in liquid, liquid is preheated to 60 DEG C, with aperture be 0.22 μm miillpore filter while hot to its into
Row filtering.Liquid pre-freeze resulting after filtering is stayed overnight, is freeze-dried to obtain 100% methacrylated GelMA material
Material.(2) spinning of gelatin aquagel fibre is oriented
The gelatin of 500mg is completely dissolved in formation gelatin solution in the HFIP of 5ml, then the solution is loaded on 5ml
It is placed on micro-injection pump in syringe, connects 15kv high-voltage electricity, parallel pole is being placed at syringe nozzle 20cm, is being put down
Row electrode spacing 3cm can form parallel orientation tunica fibrosa between electrode stem after powering on, film roll song can get cylindric
Fibre bundle, tunica fibrosa and fibre bundle are placed in 2% glutaraldehyde alcoholic solution and are crosslinked overnight.It is impregnated repeatedly in PBS liquid
After flushing, tunica fibrosa is used for cell experiment, and fibre bundle is used for zoopery.
(3) spinning of GelMA aquagel fibre is oriented
The GelMA of 500mg is dissolved in formation electrospinning liquid in the HFIP of 5ml, is protected from light 0.5g2-hydroxy-4 '-(2-
Hydroxyethoxy) -2-methylpropiophenone (photoinitiator, Irgacure 2959, Sigma-
Aldrich, St.Louis, MO) it is dissolved completely in formation photo-crosslinking liquid, electro-spinning process and gelatin in the absolute alcohol of 10ml
Aquagel fibre is similar, and electrospun fiber membrane and electrospinning fibre beam are placed in photo-crosslinking liquid middle-ultraviolet lamp and irradiate the crosslinking of completion in 30 minutes,
PBS liquid soaking flushing removes extra photocrosslinking agent repeatedly, and tunica fibrosa is used for cell experiment, and fibre bundle is used for zoopery.
Specific experiment process is shown in the preparation process and electrospinning synthesis that Fig. 1, Fig. 1 are acrylic modified gelatin GelMA
Elastic hydrogel fiber and the procedure chart for being used for cell experiment and zoopery；
The physical characteristic of 2 hydrogel scaffold of embodiment
(1) aquagel fibre shape characteristic SEM
Use SEM (SEM, S-4800；Hitachi, Tokyo, Japan) observation aquagel fibre directional characteristic and fiber
Diameter.Dry electricity spinning fibre branch is placed in drying receptacle overnight, then carries out metal spraying processing (SC7620, Quorum
Technologies, UK), SEM image sets enlargement ratio as 10000 times.Each sample respectively takes 50 different fibers at random
The measurement of diameter, arithmetic average diameter are carried out at 100 different positions using Photoshop；As a result see that Fig. 2, Fig. 2 are this hair
The SEM of the aquagel fibre of the orientation GelMA aquagel fibre of bright offer and the modified Gelatin of unused methylbenzene olefin(e) acid schemes,
Wherein, (a, d) is GelMA and Gelatin fibre image, and (c, f) is performance of the cell after fibrous framework surface is grown one day,
(b, e) is that the diameter of two kinds of fibers compares.
(2) water retention of aquagel fibre bracket
The weight of fibrous framework drying regime is denoted as W0, the weight of 24 hours after-poppets is denoted as W in soaking wateri, then will be damp
Wet fibrous framework is placed in room temperature exploitation environment, and the weight of various time points is denoted as Wt.The water retention water of different time points
Retention WR=Wt/W0* 100%, as a result see that Fig. 3, Fig. 3 are GelMA and orient the dry survey of gelatin aquagel fibre
Test result.
(3) degradation rate of aquagel fibre bracket
The degradation rate test of fibrous framework is to replace liquid in the PBS for place it in 37 DEG C and daily.Various time points take
Freeze-drying weighing out is degradation rate with former weight ratio；Fig. 4 is GelMA and orients the external degradation survey of gelatin aquagel fibre
Test result.
(4) mechanical characteristic of aquagel fibre bracket
Directional fiber film is obtained thicker tunica fibrosa using the method that superposition is collected to impregnate in PBS solution 2 hours, is cut
The sheet sample to form 15mm*3mm*0.3mm is cut, is fixed on mechanical test instrument and longitudinal stretching is carried out with 10mm/min speed,
Until break, it can be to be distributed stretching-inflection curves of two kinds of tunica fibrosas of acquisition, break length, Young's modulus.Separately take two panels phase
Same film is stretched repeatedly, and tensile elongation is the 200% of raw footage, is stretched to longest every time and is stopped 1min, obtains two kinds of fibres
Tie up the antifatigue curve of film.
Fig. 5 is GelMA and orients the mechanical experimental results of gelatin aquagel fibre, wherein (a) is mechanics survey
Examination (b) is tensile elongation curve, (c) stretches ability of anti-deformation test repeatedly, (d) is the elasticity modulus of two kinds of fibers.
Adherency growth of 3 cell of embodiment on fibrous framework
(1) form of the MSCs on bracket
MSCs kind is planted on tunica fibrosa, and culture removes culture solution after 24 hours, and PBS is rinsed three times, uses 4% paraformaldehyde
PBS is rinsed three times after fixed half an hour, then serial dehydration is carried out using the alcoholic solution for being stepped up concentration, in drying receptacle
In be dried, after metal spraying carry out SEM observation.
MSCs removes culture medium after cultivating on tunica fibrosa 24 hours, 4% paraformaldehyde fixed half an hour, PBS rinses three
Time, penetrating processing 20 minutes is carried out using 0.3% Triton X-100,4% bovine serum albumin(BSA) blocks after PBS is rinsed again
45 minutes, it finally is protected from light progress cytoskeleton fluorescent staining in 40 minutes using the phalloidine of 50mg/ml, nucleus uses DAPI
Dyeing, the fluoroscopic image of fluorescence microscope cell after PBS is rinsed three times.
(2) toxicity test LIVE/DEAD of the bracket to cell
The plant of MSCs kind removes culture medium behind on tunica fibrosa three days, is rinsed three times with PBS, then extremely dyes the work prepared
Solution (5 μ l calceins and 20 μ l ethidium persons of outstanding talent not dimer being added in 10ml PBS) is added in sample, each
200 μ l of sample is incubated at room temperature 30min, removes staining solution, and PBS is cleaned 3 times, taken pictures.
(3) proliferation experiment CCK-8 of the cell on bracket
Increasing of the test MSCs on two kinds of tunica fibrosas after 1,2,3 day is tested using cell counting kit-8 (CCK-8)
Situation is grown, PBS is cleaned three times after culture medium removal, and 300 μ L culture solutions add the CCK-8 reagent of 30 μ L, and it is small to be put into 37 ° of incubators 2
When.Then the incubation medium of 100 μ l is transferred in another 96 orifice plate.Suction is finally read under 450nm wavelength with microplate reader
The numerical value of luminosity.
(4) influence of the bracket to Hippocampal Neuron Cells form and axon growth
Hippocampal Neuron Cells are sucked out culture medium after cultivating on tunica fibrosa 24 hours, the PBS of preheating rinses three times, and 4%
Paraformaldehyde fixes half an hour, and PBS is rinsed three times, carries out penetrating processing 20 minutes, PBS using 0.3% Triton X-100
4% bovine serum albumin(BSA) blocks 2 hours after rinsing again, and specific 4 DEG C of primary antibody is added overnight, and secondary antibody is added and is incubated for one hour.
DAPI liquid is added and carries out nuclear targeting, fluorescence microscope is taken pictures.
(5) cell permeates the performance migrated on bracket
Thicker tunica fibrosa is obtained using multiple-layer stacked method when electrospinning collects directional fiber film, MSCs kind is planted in two kinds
On tunica fibrosa, after 1,2,3 day, phalloidine fluorescent staining is carried out, Laser Scanning Confocal Microscope obtains stereo-picture, and observation is thin
Born of the same parents enter the depth of tunica fibrosa, compare tunica fibrosa to the permeability of cell.
Testing result is shown in that Fig. 6~Fig. 8, Fig. 6 are cell dead test living and CCK-8 cell Proliferation vigor test result, wherein
(a) blank control group, (b) GelMA aquagel fibre group, (c) Gelatin aquagel fibre group, (d) CCK-8 cell Proliferation is surveyed
Test result, (e) cell viability compares；Fig. 7 is the fluorogram that cell is grown on tunica fibrosa, wherein (a) MSCs cell, (b) extra large
Horse neuronal cell；The cell migration that Fig. 8 Laser Scanning Confocal Microscope is shown enters internal stent depth map.
Repair of the embodiment 4GelMA elastic hydrogel fibrous framework to spinal cord
90 female sd inbred rats are randomly divided into blank control group, GelMA group, and Gelatin group is implanted into the marrow hemisection of rat chest
Defect point is repaired, and lower limb function recovery situation is evaluated, and carries out related nerve regneration, glial scars regeneration correlation, blood vessel again
Raw relevant histology.
As a result as shown in Fig. 9~11, Fig. 9 is that GelMA elastic hydrogel fibrous framework repairs result figure to mouse spinal cord,
In, A-a:GelMA aquagel fibre beam, A-b: rat spinal cord defect model, A-c: stenter to implant defect point takes after A-d:12 weeks
The spinal cord of material is from top to bottom blank control group, gelatin group control group, GelMA group.A-e-A-h be rat it is postoperative 0,4,8,
The image of 12 weeks lower limb functions evaluation.B is the protein immunization fluorescent staining of neural process axis, and C is that nestin and tuj-1 epidemic disease fluorescence contaminate
Color, D are immunofluorescence histogram.
Figure 10 is GFAP the and CSPG immunofluorescence dyeing that marker glial scar is formed.
Figure 11 is the CD31 immunofluorescence dyeing (A) and statistics histogram (B) and (C) for indicating vascular endothelial cell formation
Evaluate the BBB scoring of the postoperative lower limb function recovery situation of animal.
It is learnt from experimental result, the repairing effect of all experimental groups is better than blank control group.
Embodiment 5
Experimental summary
Result description in relation to materialogy performance:
As bionic material, it is thin to guide that the microscopic appearance structure of bracket must imitate extracellular matrix to greatest extent
Born of the same parents' sticks and grows.Neural axon fiber is in longitudinal arrangement, the transmission between brain and different segment spinal cord in spinal cord
Signal.So ideal spinal cord rack material should have with the consistent oriented structure of neural axon, to guide the life of nerve fibre
It is long to extend.By electrostatic spinning-parallel pole collection technique, GelMA the and Gelatin aquagel fibre surface light that is collected into
Sliding, a no beading can reach fine microcosmic oriented structure.The avarage fiber diameter of Gelatin is that 1.4um ± 0.13um is greater than
The average fibre diameter of GelMA is 1.1um ± 0.13um (p < 0.05).
It is compared in order to more intuitive with orientation bracket, we have prepared two kinds of hydrogels of Gelatin and GelMA simultaneously
Non-directional fiber, it can be seen that non-directional fibrous framework surface is equally smooth, and a no beading show collection mode not
The same property that will not change itself.
All GelMA and Gelatin orientation hydrogel scaffold material is demonstrated by glutinous bullet when carrying out extension test
Typical stress-inflection curves that property material has.GelMA hydrogel scaffold is demonstrated by bigger viscoelasticity, and maximum tension is long
Degree GelMA hydrogel scaffold has reached 4.54 times of raw footage, much larger than 1.94 times of Gelatin.GelMA hydrogel scaffold poplar
Family name's modulus is significantly lower than Gelatin hydrogel scaffold (p < 0.05), shows that GelMA bracket has more soft mechanical characteristic.It follows
Zernike annular polynomial-strain testing is for evaluating bracket to the compliance and hysteresis quality of external force resistance deformation, the Gelatin water after stretching repeatedly
There is apparent elasticity modulus decline (slope decline) in gel stent, and GelMA hydrogel scaffold is without substantially changeing, it was demonstrated that
GelMA has more soft and strong ability of anti-deformation advantage.
Water absorption rate is mainly used for evaluating the variation of the water content on bracket as time goes by.GelMA hydrogel scaffold fills
The water of own wt 617.4 ± 9.94% can be absorbed after sub-dip bubble, hence it is evident that be higher than Gelatin hydrogel scaffold 526 ±
11.94% (p < 0.05).As time goes by, GelMA hydrogel scaffold moisture content is consistently higher than Gelatin hydrogel scaffold,
GelMA hydrogel scaffold moisture content is 402.2 ± 20.6% after 10 days, (the p < of moisture content 343 ± 14.5% higher than Gelatin
0.05).Experiment shows the water absorption and water retention property of GelMA better than Gelatin, and the GelMA material of high-moisture can be preferably
Better nutrition and metabolism is provided for the cell in bracket to support.
Degradability is one of biologic bracket material important feature.Find that the GelMA degradation speed of photo-crosslinking is bright in experiment
The aobvious Gelatin for being faster than glutaraldehyde cross-linking, when testing the 3rd day, the surplus of GelMA hydrogel scaffold is 94.8 ± 0.84%,
The surplus of Gelatin hydrogel scaffold be 95.2 ± 0.84%, 35 days after GelMA hydrogel scaffold weight only remain 19 ±
1.58%, and Gelatin hydrogel scaffold residue 40 ± 1.58% (p < 0.05).
Result description in relation to cell experiment:
After mesenchymal stem cell is cultivated one day on fibrous framework surface, SEM observation is taken pictures, and BMSCs exists as the result is shown
On two kinds of hydrogel scaffolds, form is elongated, and adherency is good, but since GelMA hydrogel scaffold is more soft and it is preferable anti-to have
Deformability, cell migrates on GelMA hydrogel scaffold to depths, and cell still is limited on Gelatin hydrogel scaffold
Rack surface.
After BMSCs is cultivated one day on aquagel fibre bracket, phalloidine Fluorescent Staining Observation can be with from fluorescence picture
Find out, cell shown on two kinds of brackets with the consistent aligned growth mode of machine direction, form is elongated, and actin is bright
Aobvious, cell adhesion can be provided and guide its growth by showing GelMA hydrogel scaffold and Gelatin hydrogel scaffold, and be compareed
Although the actin of cell is obvious in group, disperse towards surrounding.
The biocompatibility of biological support is mainly evaluated from the dead Coloration experiment of the work of cell.GelMA and Gelatin
Hydrogel scaffold compared with the control group, only seldom dead cell.This shows that GelMA and Gelatin hydrogel scaffold can't
The death of inducing cell has good biocompatibility, and promotes the ability of cell attachment.But Gelatin and GelMA
On BMSCs it is more elongated, and adhered to towards the same direction, and it is relatively round and smooth to cultivate cellular morphology on TCP, explanation
The ability that there is GelMA and Gelatin inducing cell to grow towards the same direction.GelMA hydrogel branch after BMSC is cultivated 3 days
Cell viability on frame and Gelatin hydrogel scaffold is respectively 95 ± 3% and 89 ± 5%, be above blank control group 85 ±
6% (p < 0.05).
The height of absorbance is positively correlated with the vigor of cell in CCK-8 experiment, the OD of Control when planting the 1st day
Value be 1.35 ± 0.24, GelMA hydrogel group OD value be 1.87 ± 0.21, Gelatin hydrogel group OD value be 1.43 ±
0.18.It is 2.71 ± 0.29 that when planting the 3rd day, the OD value of Control, which is the OD value of 2.3 ± 0.22, GelMA hydrogel group,
The OD value of Gelatin hydrogel group is 2.37 ± 0.25.After plantation on the 1st day and the 3rd day GelMA hydrogel scaffold cell work
Power is obviously higher than Gelatin hydrogel scaffold and blank control group (p < 0.05).GelMA hydrogel scaffold is prompted to have good
Good biocompatibility can provide preferably growth for cell and support.
Better axon elongation condition can be provided for neuron in order to verify GelMA and Gelatin hydrogel scaffold.I
Hippocampal Neuron Cells are cultivated 1 day on four kinds of brackets after carry out Tuj-1 neuron skeleton immunofluorescence label, as a result
Prompt the growth of GelMA orientation hydrogel scaffold epineural member preferably, axon elongation longest, and Gelatin orients hydrogel scaffold
Take second place, control group neuron is sparse, and aixs cylinder is almost without extension, although refreshing on GelMA and Gelatin non-directional hydrogel scaffold
Through first well-grown, aixs cylinder can also extend, and still, aixs cylinder prolonging direction is inconsistent, be unfavorable for axon regeneration.Show
GelMA and Gelatin orientation hydrogel scaffold promotes sticking for neuron, and is conducive to the growth of neuron.
In order to quantify to compare transfer ability of the cell in two kinds of fibrous frameworks, BMSCs kind is planted in fibrous framework surface,
Respectively at progress phalloidine fluorescent staining in the 1st, 2,3 day, Laser Scanning Confocal Microscope took pictures and carries out image reconstruction (Fig. 8), from Fig. 8
As can be seen that the cell on Gelatin hydrogel scaffold does not migrate down substantially, and GelMA water with the increase of incubation time
Cell on gel stent is gradually migrating down.The depth that cell migrates downward on GelMA hydrogel scaffold is obviously big
Depth of the cell on GelMA hydrogel scaffold be up to 197.5 ± 18.1 μm when Gelatin hydrogel scaffold, third day, and
Depth on corresponding Gelatin hydrogel scaffold is 60.6 ± 8.4 μm (p < 0.05).
The description of zoopery correlated results:
9 segment spinal cord injury of chest causes lower limb disorder, may determine that bracket by evaluating lower limb function recovery situation
Repair the effect of spinal cord.The evaluation of rat hindlimb motor function is carried out using BBB scale within postoperative 1st, 2,3,4,6,8,10,12 week.
Generally speaking postoperative control group and Gelatin group rat lower limb autokinetic movement functional rehabilitation are slower and limited, and GelMA group rat is extensive
It is multiple preferable.At first 2 weeks, indifference between three groups of rats started at the 3rd week, and GelMA group and the scoring of Gelatin group are in blank
Control group has significant difference.Since the 5th week, GelMA group was significantly better than other two groups.The scoring of GelMA group reaches at 12 weeks
12.4 ± 1.67, hence it is evident that be higher than blank group 6.6 ± 1.52 and Gelatin group 8 ± 1.23 (p < 0.05).
Spinal cord nerve regeneration observation index includes the neural stem cell of anti-nestin label, anti-Tuj- in this research
The neuron of 1 label,.NSCs is migrated and is broken up to damage location under local damage stimulation, and 12 weeks remain to after injury for we
Observe the presence of NSCs in damage location, it was demonstrated that this spontaneous reparation be it is persistently existing, pass through photodensitometric quantitation measurement hair
Now show that the optical density for the cell Control group of nestin antibody label is the light of 96 ± 20, GelMA hydrogel scaffold group
Density is that the optical density of 524 ± 110, Gelatin hydrogel scaffold group is 274 ± 32.The cell of Tuj-1 antibody label
The optical density of Control group is that the optical density of 1684 ± 102, GelMA hydrogel scaffold group is 14783 ± 685, Gelatin water
The optical density of gel stent group is 11984 ± 698.NSCs is significantly more than Gelatin group and blank control group in GelMA group spinal cord
(p < 0.05), it was demonstrated that GelMA hydrogel scaffold surrounding NSCs preferably is migrated and survived into bracket.And neuron is spinal cord
The cell base of functional rehabilitation, equally, the neuron by NSCs differentiation of Tuj-1 label are obvious in GelMA hydrogel scaffold
More than other two groups (p < 0.05).
Synaptic contact is formd between neuron in order to confirm new life and completes functionalization, we use cynapse capsule
Vacuolar membrane albumen (synaptophysin) antibody marks prominent axis, and quantitative result shows that the optical density of Control group is 121 ± 24,
The optical density of GelMA hydrogel scaffold group is that the optical density of 1445 ± 128, Gelatin hydrogel scaffold group is 378 ± 45.Figure
Piece shows that the synaptophysin positive signal of GelMA hydrogel scaffold group is apparently higher than Gelatin hydrogel scaffold group and sky
White control group (p < 0.05), it was demonstrated that there is axis of more dashing forward to be formed in GelMA hydrogel scaffold.
Damage can lead to proliferation of astrocytes, then be changed into glial scars, form the physical obstacle of aixs cylinder connection.
Glial fibrillary acid protein (GFAP) is used for labeled analysis astroglia.It is marked by spectrodensitometry, GFAP antibody
The optical density of cell Control group is that the optical density of 4661 ± 520, GelMA hydrogel scaffold group is 775 ± 63, Gelatin water
The optical density of gel stent group is 2271 ± 265.CGelatin group and blank control group have found that a large amount of astroglias increase
It is raw, and control group and Gelatin group are considerably less than in GelMA group spinal cord, it was demonstrated that soft GelMA hydrogel scaffold, which has, to be inhibited
The ability that glial scars are formed.
Labeled analysis glial scars are used for using chondroitin sulfate polysaccharide (CSPG).The cell Control of CSPG antibody label
The optical density of group is that the optical density of 5046 ± 410, GelMA orientation hydrogel scaffold group is 752 ± 75, Gelatin orientation water-setting
The optical density of glue bracket group is 1029 ± 154.Gelatin group and blank control group have found that glial scars are formed, and GelMA group
Control group and Gelatin group are considerably less than in spinal cord.
By the fluorescence intensity discovery of the vascular endothelial cell marked of CD31 after comparison 12 weeks, GelMA orients hydrogel branch
There are a large amount of revascularizations in frame, hence it is evident that more than Gelatin orientation hydrogel scaffold group and blank group (p < 0.05), quantitative result
The optical density of display Control group is that the optical density of 420 ± 50, GelMA hydrogel scaffold group is 5130 ± 410, Gelatin water
The optical density of gel stent group is 1505 ± 200.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (10)
1. a kind of elastic hydrogel fiber, is obtained by acrylic modified gelatin by electrostatic spinning.
2. elastic hydrogel fiber according to claim 1, which is characterized in that in the acrylic modified gelatin
The grafting rate of methacrylic acid is 80~100%.
3. elastic hydrogel fiber according to claim 1, which is characterized in that the electrostatic spinning specifically:
1) acrylic modified gelatin solution is placed on syringe pump, carries out electrospinning, obtains tunica fibrosa or fibre bundle；
2) obtained tunica fibrosa or fibre bundle are crosslinked, obtain elastic hydrogel fiber.
4. elastic hydrogel fiber according to claim 3, which is characterized in that the acrylic modified gelatin is molten
Solvent in liquid is hexafluoroisopropanol.
5. elastic hydrogel fiber according to claim 3, which is characterized in that the design parameter of the electrospinning are as follows: 10~
20kv high-voltage electricity is placing parallel pole at 18~22cm of syringe nozzle, and parallel pole 2~4cm of spacing powers on
Afterwards with the injection of 0.05~0.15ml/min speed.
6. elastic hydrogel fiber according to claim 3, which is characterized in that the crosslinking of the step 2) is ultraviolet light friendship
Connection.
7. elastic hydrogel fiber according to claim 6, which is characterized in that the photoinitiator of the ultraviolet light cross-linking is
2- hydroxyl -4 '-(2- hydroxy ethoxy) -2- methyl phenyl ketone solution.
8. elastic hydrogel fiber according to claim 1, which is characterized in that the acrylic modified gelatin is pressed
It is obtained according to following methods preparation method:
Methacrylic acid anhydride reactant is added dropwise into gelatin solution, obtains reaction solution；
Reaction solution is dialysed, is filtered, the modified gelatin of olefin(e) acid is obtained,
Wherein, the molecular cut off of the dialysis bag filter is 12~14kDa.
9. a kind of preparation method of elastic hydrogel fiber, obtains elasticity by electrostatic spinning for acrylic modified gelatin
Aquagel fibre.
10. elastic hydrogel fiber described in a kind of claim 1~8 any one is used for the tissue of spinal cord reparation again in preparation
Application in raw bracket.
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| CN112972774A (en) * | 2019-12-13 | 2021-06-18 | 中国科学院深圳先进技术研究院 | Coaxial electrostatic spinning GelMA/PLGA-LysoGM1 and preparation method and application thereof |
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