CN105536051B - A kind of core-shell type nano fibrous framework and its method with melanocyte structure tissue engineering material - Google Patents
A kind of core-shell type nano fibrous framework and its method with melanocyte structure tissue engineering material Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Abstract
A kind of method for building tissue engineering material the invention discloses core-shell type nano fibrous framework and its with melanocyte.The wherein described core-shell type nano fiber is prepared by electrostatic spinning, and shell is biocompatible materials, and stratum nucleare is to be cross-linked in situ polyalcohol hydrogel.Tissue engineering material is built using the nano fiber scaffold and melanocyte or itself and fibroblast, keratinocyte, can be used for depigmentation disease（Such as leucoderma）Treatment.The core-shell type nano fibrous framework of the present invention has good biocompatibility and biological degradability, and the controlled release that drug loading can be used for, realize drug, growth, proliferation and the transplanting of melanocyte and co-cultured cell can be effectively supported with the tissue engineering bracket of its structure.
Description
Technical field
The invention belongs to biomedical sector more particularly to a kind of core-shell type nano fibrous frameworks with drug slow release function
And its method with melanocyte structure tissue engineering material.
Background of invention
Leucoderma is a kind of common multiple depigmentation dermatoses, and clinical manifestation is mainly that limitation or office's hair property are white
Color spot piece, clear border, no conscious sympton, histology and immunocytochemistry show that its skin lesion epidermal melanophore disappears.In vain
Purplish or white patches on the skin wind incidence in crowd reaches 0.5-2%, due to a lack of melanin at hickie, is easy even to cause canceration by sun burns, together
When due to influence human appearance image, often cause serious mental handicape to patient, reduce its quality of life.Traditional treatment
Means mainly have drug therapy and light therapy.But drug therapy and the cure rate of ultraviolet light irradiation are limited.Surgical operation therapy side
Method includes mainly AUTOEPIDERMIC GRAFTING, the transplanting of melanocyte suspension etc., and the confirmation of these therapies is effective.But epidermic grafting
It needs large area to take skin, is not suitable for large area therapy of vitiligo, and the transplanting of melanocyte suspension can be drawn materials with smaller skin, be treated
Large range of skin lesion, has a good application prospect.The main problem of the therapy is that cell suspension is in moving part hardly possible at present
Attachment, in addition graft procedure can cause to damage to cell, affect the treatment.It is raw meanwhile in melanocyte culture and migration process
The drugs such as the bio-pharmaceuticals such as the long factor and antioxidant play the role of improving melanocyte activity and transplanting curative effect well.
Therefore, suitable melanocyte carrier is built, realizes culture and the transfer integration of melanocyte, while assigning carrier there is medicine
The function of object sustained release, can effectively improve the success rate of suspension transplantation therapy.
Using tissue engineering technique, by cell loading and transfer and medicament slow release, provide new way for treatment leucoderma
Diameter.CN103046158A discloses a kind of core-shell nano fiber of sustained release dewatering medicament, and fibre fractionation has water solubility.
CN103757743A discloses a kind of core-shell nano fiber containing liposome, and active material is loaded in the liposome of stratum nucleare
To realize sustained release.CN 104337755A disclose a kind of co-axial nano drug-loading fibre film of pH sensibility, to realize gastrointestinal tract target
To administration.CN 103505758B disclose a kind of hud typed chitosan nano fiber, are used as hemostatic material.Core disclosed above
Shell nano fiber scaffold patent is used for the sustained release of drug, it is impossible to be used in the culture of human melanocyte.
Invention content
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of core-shell type nano fibrous framework and its with it is black
The method of plain cell construction tissue engineering material.
The purpose of the present invention is achieved through the following technical solutions：A kind of core-shell type nano fibrous framework, the branch
Frame is made of core-shell type nano fiber, and the shell of the core-shell type nano fiber is biocompatible materials, and stratum nucleare is controllable hands over
Connection type macromolecule hydrogel, fiber outer diameter is at 100nm-10 μm.
Further, it is prepared by following steps：
（1）Biocompatible materials are dissolved in shell solvent, the solution that weight percent is 1-10% is made into, obtains
Shell solution；The biocompatible materials are one in gelatin, collagen, fibrin, hyaluronic acid, chondroitin sulfate
Kind or it is arbitrary two kinds according to mass ratio 1:50-50:1 mixing composition；
（2）Core layer material is dissolved in stratum nucleare solvent, the solution that mass fraction is 1-10% is configured to, obtains stratum nucleare solution；
The core layer material presses 10-50 by vinyl monomer, di-vinyl monomers and stratum nucleare polymer:0.1-5:The weight ratio of 50-90
Example mixing composition；
（3）Photoinitiator is added in stratum nucleare solution, the mass fraction of photoinitiator is 0.1-3%；
（4）Shell solution and stratum nucleare solution are respectively charged into syringe respectively, spinning voltage 5-30kv, receive distance 5-
30cm, 20-50 DEG C of temperature, shell solution fltting speed 0.1-2ml/h, stratum nucleare solution are 4 with shell solution fltting speed ratio:1-
1:4, coaxial electrostatic spinning is carried out under ultraviolet lighting, and core-shell type nano fibrous framework is prepared；
（5）Core-shell type nano fibrous framework dries 2-48 hours at vacuum, 20-50oC；Then with crosslinking agent to hud typed
Nano fiber scaffold rinses cleared remaining crosslinking after crosslinking Treatment in 20-50oC crosslinking Treatments 0.5-24 hours with pure water
Agent；
（6）It is dried 2-48 hours under 30-120oC under the conditions of air blast；2-24h is dried under vacuum condition under 20 ~ 40oC,
Remove residual solvent.
Further, it is prepared by following steps：
（1）Biocompatible materials are dissolved in shell solvent, the solution that weight percent is 1-10% is made into, obtains
Shell solution；The biocompatible materials are chitosan；
（2）Core layer material is dissolved in stratum nucleare solvent, the solution that mass fraction is 1-10% is configured to, obtains stratum nucleare solution；
The core layer material presses 10-50 by vinyl monomer, di-vinyl monomers and stratum nucleare polymer:0.1-5:The weight ratio of 50-90
Example mixing composition；
（3）Photoinitiator is added in stratum nucleare solution, the mass fraction of photoinitiator is 0.1-3%；
（4）Shell solution and stratum nucleare solution are respectively charged into syringe respectively, spinning voltage 5-30kv, receive distance 5-
30cm, 20-50 DEG C of temperature, shell solution fltting speed 0.1-2ml/h, stratum nucleare solution are 4 with shell solution fltting speed ratio:1-
1:4, coaxial electrostatic spinning is carried out under ultraviolet lighting, and core-shell type nano fibrous framework is prepared；
（5）Core-shell type nano fibrous framework dries 2-48 hours at vacuum, 20-50oC；Then with crosslinking agent to hud typed
Nano fiber scaffold rinses cleared remaining crosslinking after crosslinking Treatment in 20-50oC crosslinking Treatments 0.5-24 hours with pure water
Agent；
（6）Then it uses sodium hydroxide-aqueous sodium carbonate to impregnate core-shell type nano fibrous framework 24 hours, then uses deionization
Water cleans, and in the sodium hydroxide-aqueous sodium carbonate, the quality proportioning of the gross mass and water of sodium hydroxide and sodium carbonate is 1-
10:90-99 mixing compositions, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1；
（7）It is dried 2-48 hours under 30-120oC under the conditions of air blast, dries 2-24h under vacuum condition under 20 ~ 40oC,
Remove residual solvent.
Further, the shell solvent is selected from water, formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, two
One or any two kinds by weight ratio 10 in methyl sulfoxide, dichloromethane, chloroform:1-1:The mixing of 10 mixing compositions is molten
Agent.The stratum nucleare solvent is formic acid, acetic acid, trifluoroacetic acid, water, one kind in ethyl alcohol or two kinds by weight ratio 10 arbitrary:1-
1:The mixed solvent of 10 mixing compositions.
Further, the vinyl monomer is selected from polyethylene glycol（Methyl）Acrylate,（Methyl）Acrylic acid hydroxyl second
Ester,（Methyl）Hydroxypropyl acrylate, vinyl pyrrolidone,（Methyl）Acrylic acid.The di-vinyl monomers are selected from N, N'-
Methylene-bisacrylamide, ethylene glycol two（Methyl）Acrylate, diglycol two（Methyl）Acrylate, two three second of contracting
Glycol two（Methyl）Acrylate, tetraethylene-glycol two（Methyl）Acrylate, polyethylene glycol two（Methyl）Acrylate, 1,
4- butanediol diacrylates.The stratum nucleare polymer be selected from polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, thoroughly
Bright matter acid, chondroitin sulfate.
Further, the photoinitiator is selected from 2- hydroxyl -1- [4-（Hydroxyl）Phenyl] -2- methyl-1s-acetone, phenyl
Bis- (2,4,6- trimethylbenzoyls) phosphine oxides, Benzophenone, -2 methyl of 1- [4- (2- hydroxyl-oxethyls) phenyl] -2- hydroxyls -
1- propane -1- ketone, 1- hydroxycyclohexyl phenyl ketones.
Further, the crosslinking agent is selected from sodium sulphate, sodium citrate, sodium tripolyphosphate, glutaraldehyde, glyoxal, water
Poplar aldehyde, Geniposide, vanillic aldehyde etc..
A kind of method of core-shell type nano fibrous framework and melanocyte structure tissue engineering material, this method is that will go out
Treated that core-shell type nano fibrous framework is placed in tissue culture plate for bacterium, to tissue culture plate in inject culture solution, be placed in thin
In 37oC, the CO that volume fraction is 5% in born of the same parents' incubator2, saturated humidity condition hatched；It will be by 1-40: 0-200: 0-
Melanocyte, fibroblast and the keratinocyte that 100 number of cells ratio co-cultures are with 5 × 103-5×105 It is a thin
Born of the same parents/cm2Density be seeded on the core-shell type nano fibrous framework hatched, while 1-10 milliliters of cell culture fluid is added,
Cell incubator is placed in 37oC, the CO that volume fraction is 5%2, cultivate under the conditions of saturated humidity, replace a cell within every two days
Culture solution, incubation time are 1-10 days, can get the composite material of cell-core-shell type nano fibrous framework.
Further, the culture solution by 100 milliliters of F12 culture mediums, 0.1-100 milliliters of FBS, 10-50000 nanogram CT,
The ingredients mixing groups such as 10-50000 microgram IBMX, 0.1-100 milliliter Glutamine and 100-500000 micrograms Gentamicin
At.
The beneficial effects of the invention are as follows：There is the nanofiber of gained nucleocapsid, nuclear material to use cross-linking type water-setting
Glue can efficiently control load and its rate of release of drug by the design and customization of hydrogel crosslink density.Nanofiber
Holder has porous structure, is suitble to cell attachment, growth.Simultaneously there is good mechanical performance, preparation, load, transfer and
Migration process does not occur broken and is broken, and meets the requirement of transfer and transplanting.The structure of cell-nano fiber scaffold composite material
In the process, form of the cell on biocompatibility nano fiber scaffold material is good, and porous nanofibrous structures are beneficial to training
The update of nutrient solution makes cell keep good activity to promote the proliferation of cell.
Description of the drawings
Fig. 1 is the structural schematic diagram of core-shell type nano fiber of the present invention, in figure, shell 1, stratum nucleare 2.
Specific implementation mode
The invention belongs to biomedical sectors, have good biocompatibility core-shell nano fine using tissue engineering technique structure
Dimensional scaffold material, drug can be carried on its stratum nucleare, and realize the controlled release of drug.The timbering material for melanocyte or its
With fibroblast, keratinocyte（Altogether）Culture, realizes in vitro culture and the transplanting of cell.More particularly to nucleocapsid
The preparation of nano fiber scaffold material, the structure of cell-nano fiber scaffold composite material and its application, meet depigmentation disease
（Such as leucoderma）The requirement adjusted with skin color.
The present invention selects the good material of biocompatibility, nano fiber scaffold material preparation mild condition porous to receive
Rice fibre structure imitates human body cell epimatrix, can promote the attaching, growth and proliferation of cell, and cell is made to keep good work
Property.There is the nano fiber scaffold material of crosslinked processing superior mechanical performance, satisfaction can directly be taken from tissue culture plate
Go out the requirement shifted and transplanted for cell.Nano fiber scaffold material can carrying medicament, and can pass through that adjust crosslink density real
The controlled release of existing drug, improves the curative effect in melanocyte activity and migration process in incubation well.In addition, this
Invention keeps cell activity by inoculum density, incubation time and the cultural method of adjusting cell, realizes to Different Individual skin
The regulation and control of color, the expression that the effect of property that makes melanocyte is succeeded in transplanting have actual operability, are big face
The adjusting of product depigmentation disease and skin color provides a great convenience.
A kind of core-shell type nano fibrous framework of the present invention, is prepared by following steps：
Biocompatible materials are dissolved in shell solvent, the solution that weight percent is 1-10% is made into, obtains shell
Solution；
Core layer material is dissolved in stratum nucleare solvent, the solution that mass fraction is 1-10% is configured to, obtains stratum nucleare solution；Institute
Core layer material is stated by vinyl monomer, di-vinyl monomers and stratum nucleare polymer by 10-50:0.1-5:The weight ratio of 50-90
Mixing composition；
Photoinitiator is added in stratum nucleare solution, the mass fraction of photoinitiator is 0.1-3%；
Shell solution and stratum nucleare solution are respectively charged into syringe respectively, spinning voltage 5-30kv, receive distance 5-
30cm, 20-50 DEG C of temperature, shell solution fltting speed 0.1-2ml/h, stratum nucleare solution are 4 with shell solution fltting speed ratio:1-
1:4, coaxial electrostatic spinning is carried out under ultraviolet lighting, and core-shell type nano fibrous framework is prepared；
Core-shell type nano fibrous framework dries 2-48 hours at vacuum, 20-50oC；Then with crosslinking agent to core-shell type nano
Fibrous framework rinses cleared remaining crosslinking agent after crosslinking Treatment in 20-50oC crosslinking Treatments 0.5-24 hours with pure water（It hands over
The specific implementation means of the quality proportioning and crosslinking Treatment that join agent and core-shell type nano fibrous framework are the known normal of this field
Know, for example, Journal of Biomedical Materials Research Part A 2012,100A can be referred to,
673-683.）；
Then as needed, impregnated 24 hours with sodium hydroxide-aqueous sodium carbonate, then cleaned with deionized water（Generally
The PH of solution of the cleaning to after cleaning is neutrality）, in the sodium hydroxide-aqueous sodium carbonate, sodium hydroxide and sodium carbonate
Gross mass and the quality proportioning of water are 1-10:90-99 mixing compositions, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:
1；It is dried 2-48 hours under 30-120oC under the conditions of air blast again；Vacuum drying removal residual solvent 2-24h.
The shell solvent be selected from water, formic acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide (DMSO),
One or any two kinds by weight ratio 10 in dichloromethane, chloroform:1-1:The mixed solvent of 10 mixing compositions.The core
Layer solvent is formic acid, acetic acid, trifluoroacetic acid, water, one kind in ethyl alcohol or two kinds by weight ratio 10 arbitrary:1-1:10 mixing groups
At mixed solvent.
The biocompatible materials are by chitosan, gelatin, collagen, fibrin, hyaluronic acid, chondroitin sulfate
One kind in element or it is arbitrary two kinds according to mass ratio 1:50-50:1 mixing composition.
The vinyl monomer is selected from polyethylene glycol（Methyl）Acrylate,（Methyl）Hydroxy-ethyl acrylate,（Methyl）
Hydroxypropyl acrylate, vinyl pyrrolidone,（Methyl）Acrylic acid.The di-vinyl monomers are selected from N, N'- di-2-ethylhexylphosphine oxides
Acrylamide, ethylene glycol two（Methyl）Acrylate, diglycol two（Methyl）Acrylate, triethylene-glycol two
（Methyl）Acrylate, tetraethylene-glycol two（Methyl）Acrylate, polyethylene glycol two（Methyl）Acrylate, Isosorbide-5-Nitrae-fourth two
Alcohol diacrylate.The stratum nucleare polymer be selected from polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, hyaluronic acid,
Chondroitin sulfate.
The photoinitiator is selected from 2- hydroxyl -1- [4-（Hydroxyl）Phenyl] -2- methyl-1s-acetone, the bis- (2,4,6- of phenyl
Trimethylbenzoyl) phosphine oxide, Benzophenone, 1- [4- (2- hydroxyl-oxethyls) phenyl] -2 methyl-1s of -2- hydroxyls-propane -1-
Ketone, 1- hydroxycyclohexyl phenyl ketones.
The crosslinking agent is selected from sodium sulphate, sodium citrate, sodium tripolyphosphate, glutaraldehyde, glyoxal, salicylide, capital Buddhist nun
Flat, vanillic aldehyde etc..
The side of cell-core-shell type nano fibrous framework tissue engineering material is built using above-mentioned core-shell type nano fibrous framework
Method is as follows：
By core-shell type nano fibrous framework and melanocyte for building tissue engineering material, specifically, by sterilization treatment
Core-shell type nano fibrous framework afterwards is placed in tissue culture plate, to tissue culture plate in inject culture solution, be placed in cell culture
Hatched in 37oC, the CO2 that volume fraction is 5%, saturated humidity condition in case；It will be by 1-40: 0-200:0-100's is thin
Melanocyte, fibroblast and the keratinocyte that born of the same parents' number ratio co-cultures are with 5 × 103-5×105A cell/cm2
Density be seeded on the core-shell type nano fibrous framework hatched, while 1-10 milliliters of cell culture fluid is added, is placed in thin
Born of the same parents' incubator is cultivated under the conditions of 37oC, the CO2 that volume fraction is 5%, saturated humidity, replaces a cell culture fluid within every two days,
Incubation time is 1-10 days, can get the composite material of cell-core-shell type nano fibrous framework.
The culture solution is micro- by 100 milliliters of F12 culture mediums, 0.1-100 milliliters of FBS, 10-50000 nanograms CT, 10-50000
The ingredients such as gram IBMX, 0.1-100 milliliters of Glutamine and 100-500000 micrograms Gentamicin mixing composition.
The present invention is described in detail below according to embodiment, the objects and effects of the present invention will be more apparent..Ying Li
Solution, these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
The preparation of 1. chitosans of embodiment-polyethylene oxide core-shell nano fiber scaffold material
A. by chitosan trifluoroacetic acid/dichloromethane weight ratio 3:1 mixed solvent dissolves, and is made into the shell of a concentration of 5 %
Layer solution；
B. by polyethylene oxide, polyethylene glycol methacrylate-styrene polymer, polyethylene glycol dimethacrylate, by 80：20:1
Weight ratio is dissolved in water, and is made into a concentration of 5% stratum nucleare solution, and 1% photoinitiator 1- [4- (2- hydroxyls are added before spinning
Ethyoxyl) phenyl] -2 methyl-1s of -2- hydroxyls-propane -1- ketone；
C. ultraviolet voltage is 15KV, to promote rate be 0.5ml/h to nucleocapsid, receives distance is 15cm, temperature 30oC
Electrostatic spinning is carried out under the conditions of light irradiation；
D. core-shell nano fibrous framework is dried 12 hours at vacuum, 50oC；
E.20oC under, core-shell nano fibrous framework is crosslinked 2 hours with sodium sulphate, cleared with pure water flushing after crosslinking Treatment
Remaining crosslinking agent；
F. sodium hydroxide-aqueous sodium carbonate is used（The quality proportioning of the gross mass and water of sodium hydroxide and sodium carbonate is 1:
90 mixing compositions, and the quality proportioning of sodium hydroxide and sodium carbonate is 3:1）Impregnate core-shell type nano fibrous framework 24 hours, then
PH=7.2-7.4 of the solution to cleaning is cleaned with deionized water
G. chitosan nano fiber branch is placed in convection oven, it is 24 hours dry under the conditions of 50oC；Again in vacuum item
2-24h is dried under part under 20 ~ 40oC, removes residual solvent.
H. chitosan-polyethylene oxide core-shell nano the fiber scaffold material that can be used for cell culture is obtained.
The preparation of 2. chitosans of embodiment-polyvinylpyrrolidone core-shell nano fiber scaffold material
A. by chitosan acetic acid/water（Weight proportion 1:1）Mixed solvent dissolve, be made into a concentration of 2% chitosan it is molten
Liquid；
B. by polyvinylpyrrolidone, hydroxyethyl methacrylate, N, N'- methylene-bisacrylamides, by 70：30:3 weights
Amount ratio is dissolved in water, and is made into a concentration of 9% stratum nucleare solution, and 3% photoinitiator phenyl bis- (2,4,6- tri- is added before spinning
Methyl benzoyl) phosphine oxide；
C. voltage is 20KV, shell propulsion rate is 0.5ml/h, stratum nucleare fltting speed is 1ml/h, reception distance is
18cm, temperature 20oC carry out electrostatic spinning under ultraviolet irradiation condition；
D. chitosan/polyethylene oxide-polyvinylpyrrolidone core-shell nano fibrous framework is dried 24 at vacuum, 25oC
Hour；
E.20oC under, chitosan-polyvinylpyrrolidone core-shell nano fibrous framework genipin cross-linked 12 hours, crosslinking
After processing cleared remaining crosslinking agent is rinsed with pure water；
F. sodium hydroxide-aqueous sodium carbonate is used（The quality proportioning of the gross mass and water of sodium hydroxide and sodium carbonate is 1：9
Mixing composition, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3）It impregnates core-shell type nano fibrous framework 24 hours, then uses
Deionized water cleans PH=7.2-7.4 of the solution to cleaning
G. chitosan-polyvinylpyrrolidone core-shell nano fibrous framework is placed in convection oven, is done under the conditions of 50oC
Dry 24 hours；2-24h is dried under vacuum condition under 20 ~ 40oC, removes residual solvent.
H. chitosan-polyvinylpyrrolidone core-shell nano the fiber scaffold material that can be used for cell culture is obtained.
The preparation of 3. gelatin of embodiment-polyvinyl alcohol core-shell nano fiber scaffold material
A. by gelatin trifluoroethanol/water（Mass ratio 8:2）Solvent dissolves, and is made into a concentration of 10% gelatin solution；
B. by polyvinyl alcohol, methacrylic acid, tirethylene glycol dimethylacrylate, by 60:40:0.4 weight ratio is molten
Solution is in acetic acid/water（Mass ratio 1:1）In, it is made into a concentration of 3% stratum nucleare solution, 0.2% photoinitiator 2- is added before spinning
Hydroxyl -1- [4-（Hydroxyl）Phenyl] -2- methyl-1s-acetone；
C. voltage is 25KV, shell propulsion rate is 1ml/h, stratum nucleare propulsion rate is 0.5ml/h, receiving distance is
20cm, temperature 40oC carry out electrostatic spinning under ultraviolet irradiation condition；
D. gelatin-polyethylene glycol nano fiber scaffold is dried 24 hours at vacuum, 25oC；
E.50oC under, gelatin-polyvinyl alcohol core-shell nano fibrous framework glutaraldehyde cross-linking 12 hours is used after crosslinking Treatment
Pure water rinses cleared remaining crosslinking agent；
F. gelatin-polyvinyl alcohol core-shell nano fibrous framework is placed in convection oven, it is 24 hours dry under the conditions of 50oC；
2-24h is dried under vacuum condition under 20 ~ 40oC, removes residual solvent.
G. gelatin-polyvinyl alcohol core-shell nano the fiber scaffold material that can be used for cell culture is obtained.
Embodiment 4. loads the system of chitosan-polyvinylpyrrolidone core-shell nano fiber scaffold material of bio-pharmaceutical
It is standby
A. by chitosan trifluoroacetic acid：Chloroform（Volume proportion 7:3）Mixed solvent dissolves, and the shell for being made into a concentration of 4% gathers
Sugar juice；
B. by polyvinylpyrrolidone, hydroxyethyl methacrylate, N, N'- methylene-bisacrylamides, by 70：30:3 weights
Amount ratio is dissolved in water, and is made into a concentration of 6% stratum nucleare solution, and 3% photoinitiator phenyl bis- (2,4,6- tri- is added before spinning
Methyl benzoyl) phosphine oxide and 0.1% hepatocyte growth factor；
C. voltage is 20KV, shell propulsion rate is 0.5ml/h, stratum nucleare fltting speed is 1ml/h, reception distance is
18cm, temperature 20oC carry out electrostatic spinning under ultraviolet irradiation condition；
D. chitosan-polyvinylpyrrolidone core-shell nano fibrous framework is dried 24 hours at vacuum, 25oC；
E.20oC under, chitosan-polyvinylpyrrolidone core-shell nano fibrous framework genipin cross-linked 12 hours, crosslinking
After processing cleared remaining crosslinking agent is rinsed with pure water；
F. sodium hydroxide-aqueous sodium carbonate is used（The quality proportioning of the gross mass and water of sodium hydroxide and sodium carbonate is 1：
20 mixing compositions, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:1）Impregnate core-shell type nano fibrous framework 24 hours, then
PH=7.2-7.4 of the solution to cleaning is cleaned with deionized water.
G. chitosan-polyvinylpyrrolidone core-shell nano fibrous framework is placed in convection oven, is done under the conditions of 50oC
Dry 24 hours；2-24h is dried under vacuum condition under 20 ~ 40oC, removes residual solvent.
H. the load for obtaining can be used for cell culture has chitosan/polyethylene oxide-polyethylene of hepatocyte growth factor
Pyrrolidones core-shell nano fiber scaffold material.
The preparation of the chitosan of 5. carrying medicament of embodiment-polyvinylpyrrolidone core-shell nano fiber scaffold material
A. by chitosan trifluoroacetic acid/dichloromethane（Weight proportion 3:1）Mixed solvent dissolves, and is made into a concentration of 4%
Chitosan solution；
B. by polyvinylpyrrolidone, hydroxyethyl methacrylate, N, N'- methylene-bisacrylamides, by 70：30:3 weights
Amount ratio is dissolved in water, and is made into a concentration of 6% stratum nucleare solution, and 3% photoinitiator phenyl bis- (2,4,6- tri- is added before spinning
Methyl benzoyl) phosphine oxide and 5% Quercetin；
C. voltage is 20KV, shell propulsion rate is 0.5ml/h, stratum nucleare fltting speed is 1ml/h, reception distance is
18cm, temperature 20oC carry out electrostatic spinning under ultraviolet irradiation condition；
D. chitosan-polyvinylpyrrolidone core-shell nano fibrous framework is dried 24 hours at vacuum, 25oC；
E.20oC under, chitosan-polyvinylpyrrolidone core-shell nano fibrous framework genipin cross-linked 12 hours, crosslinking
After processing cleared remaining crosslinking agent is rinsed with pure water；
F. sodium hydroxide-aqueous sodium carbonate is used（The quality proportioning of the gross mass and water of sodium hydroxide and sodium carbonate is 1：
15 mixing compositions, and the quality proportioning of sodium hydroxide and sodium carbonate is 2:3）Impregnate core-shell type nano fibrous framework 24 hours, then
PH=7.2-7.4 of the solution to cleaning is cleaned with deionized water.
G. chitosan/polyethylene oxide-polyvinylpyrrolidone core-shell nano fibrous framework is placed in convection oven, 50
It is 24 hours dry under the conditions of oC；2-24h is dried under vacuum condition under 20 ~ 40oC, removes residual solvent.
The load for obtaining can be used for cell culture has chitosan/polyethylene oxide-polyvinylpyrrolidone core of Quercetin
Shell nano fiber scaffold material.
The structure of 6. cells of embodiment-core-shell nano fibrous framework composite material
A. the chitosan described in example 1-polyethylene oxide nano fiber scaffold is placed in the alcohol that volume fraction is 75%
It is rinsed well with PBS after impregnating 12 hours；
B. it is small the tissue culture plate for covering chitosan-polyethylene oxide nano fiber scaffold to be placed in irradiation 12 under ultraviolet lamp
When；
C. 3 milliliters of cell culture fluids are injected in tissue culture plate, are placed in cell incubator in 37oC, volume fraction 5%
CO2, hatch 4 hours under saturated humidity；
D. by melanocyte with 8 × 104A cell/cm2Density be seeded on chitosan nano fiber holder, be placed in thin
Born of the same parents' incubator is in 37oC, the CO that volume fraction is 5%2, cultivate under saturated humidity；
E. a cell culture fluid is changed within every two days, incubation time is 6 days；
F. melanocyte and chitosan-polyethylene oxide nano fiber scaffold composite material are obtained, can be further used for moving
It plants.
The structure of 7. cells of embodiment-core-shell nano fibrous composite
A. chitosan/polyethylene oxide described in example 2-polyvinylpyrrolidone core-shell nano fibrous framework is placed in body
It is rinsed well with PBS after being impregnated 12 hours in the alcohol that fraction is 75%；
B. the tissue culture plate of chitosan/polyethylene oxide-polyvinylpyrrolidone core-shell nano fibrous framework will be covered
It is placed under ultraviolet lamp and irradiates 12 hours；
C. 2 milliliters of cell culture fluids are injected in tissue culture plate, are placed in cell incubator in 37oC, volume fraction 5%
CO2, hatch 4 hours under saturated humidity；
D. by melanocyte and fibroblast with ratio for 1:2, density is 8 × 104A cell/cm2It is poly- to be seeded in shell
On sugar/polyethylene oxide-polyvinylpyrrolidone core-shell nano fibrous framework, it is placed in cell incubator 37oC, volume fraction is
5% CO2, cultivate under saturated humidity；
E. a cell culture fluid is changed within every two days, incubation time is 1-10 days；
F. it obtains melanocyte and fibroblast-chitosan/polyethylene oxide-polyvinylpyrrolidone core-shell nano is fine
Composite material is tieed up, can be further used for transplanting.
The structure of 8. cells of embodiment-core-shell nano fibrous framework composite material
A. the gelatin described in example 3-polyvinyl alcohol core-shell nano fibrous framework is placed in the alcohol that volume fraction is 75%
It is rinsed well with PBS after impregnating 12 hours；
B. it is small the tissue culture plate for covering gelatin-polyvinyl alcohol core-shell nano fibrous framework to be placed in irradiation 12 under ultraviolet lamp
When；
C. 2 milliliters of cell culture fluids are injected in tissue culture plate, are placed in cell incubator in 37oC, volume fraction 5%
CO2, hatch 4 hours under saturated humidity；
D. by melanocyte, fibroblast, keratinocyte ratio with 1:2:1, density is 8 × 104A cell/cm2
It is seeded on gelatin-polyvinyl alcohol core-shell nano fibrous framework, is placed in cell incubator in 37oC, the CO that volume fraction is 5%2、
It is cultivated under saturated humidity；
E. a cell culture fluid is changed within every two days, incubation time is 6 days；
It is multiple with fibroblast/keratinocyte-gelatin-polyvinyl alcohol core-shell nano fibrous framework to obtain melanocyte
Condensation material can be further used for transplanting.
The structure of 9. cells of embodiment-core-shell nano fibrous composite
A., load described in example 4 is had to chitosan/polyethylene oxide-polyvinylpyrrolidone of hepatocyte growth factor
Core-shell nano fibrous framework be placed in volume fraction be 75% alcohol in impregnate 12 hours after rinsed well with PBS；
B. the tissue culture plate of chitosan/polyethylene oxide-polyvinylpyrrolidone core-shell nano fibrous framework will be covered
It is placed under ultraviolet lamp and irradiates 12 hours；
C. 2 milliliters of cell culture fluids are injected in tissue culture plate, are placed in cell incubator in 37oC, volume fraction 5%
CO2, hatch 4 hours under saturated humidity；
D. by melanocyte and fibroblast with ratio for 1:2, density is 8 × 104A cell/cm2It is poly- to be seeded in shell
On sugar/polyethylene oxide-polyvinylpyrrolidone core-shell nano fibrous framework, it is placed in cell incubator 37oC, volume fraction is
5% CO2, cultivate under saturated humidity；
E. a cell culture fluid is changed within every two days, incubation time is 1-10 days；
F. melanocyte and fibroblast and chitosan/polyethylene oxide-polyvinylpyrrolidone core-shell nano are obtained
Fibrous composite, melanocyte proliferation and activity are substantially better than embodiment 7.The film composite material of the load cells can be into one
Step is for transplanting.The holder of the present invention can further increase the proliferation and activity of melanocyte by carrying medicine, after being conducive to transplanting
The secondary color of focal zone improves success rate.
The present invention realizes melanocyte or the body of itself and fibroblast and keratinocyte using tissue engineering technique
Outer culture, load and transplanting.By the way that cell-core-shell nano fibrous framework composite implantation at depigmenting skin wound, is made
Melanocyte or its skin wounds are moved to by carrier material with fibroblast and keratinocyte, for skin at decoloration
Melanocyte is provided, to realize depigmentation disease（Such as leucoderma）With the adjusting of skin color.Utilize core-shell nano fiber branch
The drug loading of frame and the function of controlled release improve melanocyte activity and transplanting curative effect.
Claims (8)
1. a kind of core-shell type nano fibrous framework, the holder are made of core-shell type nano fiber, the core-shell type nano fiber
Shell be biocompatible materials, stratum nucleare be Controllable cross-linking type macromolecule hydrogel, fiber outer diameter is at 100nm-10 μm；It is special
Sign is, is prepared by following steps：
（1）Biocompatible materials are dissolved in shell solvent, the solution that weight percent is 1-10% is made into, obtains shell
Solution；The biocompatible materials be gelatin, collagen, fibrin, hyaluronic acid, one kind in chondroitin sulfate or
Arbitrary two kinds according to mass ratio 1:50-50:1 mixing composition；
（2）Core layer material is dissolved in stratum nucleare solvent, the solution that mass fraction is 1-10% is configured to, obtains stratum nucleare solution；It is described
Core layer material presses 10-50 by vinyl monomer, di-vinyl monomers and stratum nucleare polymer:0.1-5:The weight ratio of 50-90 is mixed
It is combined into；
（3）Photoinitiator is added in stratum nucleare solution, the mass fraction of photoinitiator is 0.1-3%；
（4）Shell solution and stratum nucleare solution are respectively charged into syringe respectively, spinning voltage 5-30kv, receive distance 5-
30cm, 20-50 DEG C of temperature, shell solution fltting speed 0.1-2ml/h, stratum nucleare solution are 4 with shell solution fltting speed ratio:1-
1:4, coaxial electrostatic spinning is carried out under ultraviolet lighting, and core-shell type nano fibrous framework is prepared；
（5）Core-shell type nano fibrous framework dries 2-48 hours at vacuum, 20-50oC；Then with crosslinking agent to core-shell type nano
Fibrous framework rinses cleared remaining crosslinking agent after crosslinking Treatment in 20-50oC crosslinking Treatments 0.5-24 hours with pure water；
（6）It is dried 2-48 hours under 30-120oC under the conditions of air blast；Dry 2-24h, removal under 20 ~ 40oC under vacuum condition
Residual solvent；
Or, being prepared by following steps：
（1）Biocompatible materials are dissolved in shell solvent, the solution that weight percent is 1-10% is made into, obtains shell
Solution；The biocompatible materials are chitosan；
（2）Core layer material is dissolved in stratum nucleare solvent, the solution that mass fraction is 1-10% is configured to, obtains stratum nucleare solution；It is described
Core layer material presses 10-50 by vinyl monomer, di-vinyl monomers and stratum nucleare polymer:0.1-5:The weight ratio of 50-90 is mixed
It is combined into；
（3）Photoinitiator is added in stratum nucleare solution, the mass fraction of photoinitiator is 0.1-3%；
（4）Shell solution and stratum nucleare solution are respectively charged into syringe respectively, spinning voltage 5-30kv, receive distance 5-
30cm, 20-50 DEG C of temperature, shell solution fltting speed 0.1-2ml/h, stratum nucleare solution are 4 with shell solution fltting speed ratio:1-
1:4, coaxial electrostatic spinning is carried out under ultraviolet lighting, and core-shell type nano fibrous framework is prepared；
（5）Core-shell type nano fibrous framework dries 2-48 hours at vacuum, 20-50oC；Then with crosslinking agent to core-shell type nano
Fibrous framework rinses cleared remaining crosslinking agent after crosslinking Treatment in 20-50oC crosslinking Treatments 0.5-24 hours with pure water；
（6）Then sodium hydroxide-aqueous sodium carbonate is used to impregnate core-shell type nano fibrous framework 24 hours, then clear with deionized water
It washes, in the sodium hydroxide-aqueous sodium carbonate, the quality proportioning of the gross mass and water of sodium hydroxide and sodium carbonate is 1-10:
90-99 mixing compositions, and the quality proportioning of sodium hydroxide and sodium carbonate is 1:3-3:1；
（7）It is dried 2-48 hours under 30-120oC under the conditions of air blast, dry 2-24h, removal under 20 ~ 40oC under vacuum condition
Residual solvent.
2. core-shell type nano fibrous framework according to claim 1, which is characterized in that the shell solvent is selected from water, first
It is one or any in acid, acetic acid, trifluoroacetic acid, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide (DMSO), dichloromethane, chloroform
Two kinds by weight ratio 10:1-1:The mixed solvent of 10 mixing compositions；The stratum nucleare solvent be formic acid, acetic acid, trifluoroacetic acid,
One kind or two kinds by weight ratio 10 arbitrary in water, ethyl alcohol:1-1:The mixed solvent of 10 mixing compositions.
3. core-shell type nano fibrous framework according to claim 1, which is characterized in that the vinyl monomer is selected from poly-
Ethylene glycol（Methyl）Acrylate,（Methyl）Hydroxy-ethyl acrylate,（Methyl）Hydroxypropyl acrylate, vinyl pyrrolidone,（First
Base）Acrylic acid；The di-vinyl monomers are selected from N, N'- methylene-bisacrylamides, ethylene glycol two（Methyl）Acrylate,
Diglycol two（Methyl）Acrylate, triethylene-glycol two（Methyl）Acrylate, tetraethylene-glycol two（First
Base）Acrylate, polyethylene glycol two（Methyl）Acrylate, 1,4-butanediol diacrylate；The stratum nucleare polymer choosing
From polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, hyaluronic acid, chondroitin sulfate.
4. core-shell type nano fibrous framework according to claim 1, which is characterized in that the photoinitiator is selected from 2- hydroxyls
Base -1- [4-（Hydroxyl）Phenyl] -2- methyl-1s-acetone, bis- (2,4,6- trimethylbenzoyls) phosphine oxides of phenyl, Benzophenone,
1- [4- (2- hydroxyl-oxethyls) phenyl] -2 methyl-1s of -2- hydroxyls-propane -1- ketone, 1- hydroxycyclohexyl phenyl ketones.
5. core-shell type nano fibrous framework according to claim 1, which is characterized in that the crosslinking agent is selected from sulfuric acid
Sodium, sodium citrate, sodium tripolyphosphate, glutaraldehyde, glyoxal, salicylide, Geniposide, vanillic aldehyde.
6. a kind of method of core-shell type nano fibrous framework described in claim 1 and melanocyte structure tissue engineering material,
It is characterized in that, this method is：Core-shell type nano fibrous framework after sterilization treatment is placed in tissue culture plate, is trained to cell
It supports and injects culture solution in plate, be placed in cell incubator in 37oC, the CO that volume fraction is 5%2, saturated humidity condition incubated
Change；It will be by 1-40: 0-200:Melanocyte, fibroblast and the cutin that the number of cells ratio of 0-100 co-cultures are formed
Cell is with 5 × 103-5×105 A cell/cm2Density be seeded on the core-shell type nano fibrous framework hatched, simultaneously plus
The cell culture fluid for entering 1-10 milliliters is placed in cell incubator in 37oC, the CO that volume fraction is 5%2, under the conditions of saturated humidity
A cell culture fluid is replaced in culture for every two days, and incubation time is 1-10 days, can get cell-core-shell type nano fibrous framework
Composite material.
7. according to the method described in claim 6, it is characterized in that, the culture solution is by 100 milliliters of F12 culture mediums, 0.1-100
Milliliter FBS, 10-50000 nanogram CT, 10-50000 microgram IBMX, 0.1-100 milliliter Glutamine and 100-500000 micrograms
Gentamicin mixing compositions.
8. a kind of tissue engineering material of claim 6 the method structure.
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| CN106676754A (en) * | 2016-12-30 | 2017-05-17 | 国家纳米科学中心 | Nanofiber membrane and preparation method and application thereof |
| IT201700036930A1 (en) * | 2017-04-04 | 2018-10-04 | Silk Biomaterials S R L | System that includes hydrogels and nanofibres |
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