CN104491914B - A kind of porous pluralgel-nanofiber oxygen flow dressing and preparation method thereof - Google Patents
A kind of porous pluralgel-nanofiber oxygen flow dressing and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of porous pluralgel-nanofiber oxygen flow dressing and preparation method thereof, collagen solution is adopted to obtain collagen protein nano fibrous membrane by electrospinning process, then carry out chemical crosslinking with the macromolecule polysaccharide of chitosan Yu two kinds of Nantural non-toxics of alginic acid and prepare pluralgel, mandruka-nanofiber double-layer composite material is prepared again through lyophilization mode, it is sponge state during the gel drying of dressing upper strata, it it is gel state after moisture absorption, the present invention is when without any toxic chemical cross-linking agent, prepare biological wound-surface dressing, technique is simple, safety is good, the feature of biological wound dressing prepared by the present invention is to have excellent oxygen permeability, water absorption, water penetration, poisture-penetrability and good biocompatibility, wound surface physiology moist environment can be kept, wound healing and effectively stopping blooding, wound infection can be prevented and accelerate recovery from illness, wound surface effectively can be repaired, suitable in wound, burn, the multiple wound surface such as ulcer。
Description
Technical field
The invention belongs to natural macromolecular material and biology medical material technical field, relate to one and belong to Medical wounded surface dressing, particularly to a kind of porous pluralgel-nanofiber oxygen flow dressing and preparation method thereof。
Background technology
Closed negative pressure drainage (Vacuum-assistedclosure, VAC) technology is as the new technique of a kind of Wound treating, effectively control with it and alleviate infection, accelerate wound healing, reduce the significant advantages such as medical personnel's workload and be widely used in clinical treatment, these section office started the cure mechanism correlational study of VAC technology and achieve a collection of original result of study from 1998, it has been observed that application VAC treatment can make wound surface environment be continuously in hypoxia, there are some researches show, although in wound healing early process, anaerobic environment is conducive to wound surface angeogenesis, but it is as the carrying out of wound healing, increasing tissue oxygen supply can make local cellular proliferation accelerate, tissue repairing ability strengthens, wound surface telangiectasis can be made simultaneously, resistance of blood flow reduces, blood flow rate is accelerated, tissue oozes out minimizing, swelling alleviates, thus accelerating wound healing (oxygen, ozone and the negative pressure drainage use in conjunction therapeutical effect to wound surface, JTraumaSurg, 2011, 13 (3))。For improving this present situation of wound surface low-oxygen environment, we attempt adopting oxygen delivery technique to carry out VAC treatment, and show that local oxygen supply can promote the preliminary experimental results of wound repair, and this facilitation is likely to relevant with bacteria growing inhibiting with the microcirculation disturbance improving wound tissue。
The dressing used for VAC technology at present is studied relatively fewer, early stage researcher is attempted using Cotton Gossypii, gauze etc. as medical dressing, but these dressing also exist obvious deficiency: traditional drugs cotton cloth exists when treating skin wound that healing stage is longer, wound cicatrix substantially, the process of changing dressings cause the drawback of patient suffering, gauze can cause the environment that wound surface dries more, make wound surface dehydration, cause incrustation。And the epithelization of wound surface is had obvious obstacle (development of collagen/chitosan composite table skin growth factor thrombin sponge dressing, ACAD J GCP, 2004,20 (5)) by the incrustation of wound surface。Usual hydrogel wound dressing has absorption wound fluid, keep wound surface cleaning moistening, reduce cicatrization, the advantages such as wound healing, but not there is antibiotic property, existing defects in preventing wound infection, while this just requires that often more change dressings brings considerable distress to patient, too increase the workload of medical personnel, recent dressing is then main with medical pluralgel (polyurethane, polyvinyl alcohol etc.) it is main, VAC technology can form a kind of shearing force in medical foam and wound interface, this mechanical stress is considered there is the effect promoting granulation tissue growth and angiogenesis。But, current existing medical plural gel dressing cannot realize the conduction of this stress preferably, therefore develop the Medical dressing for skin wound having excellent oxygen permeability, bacteriostasis property and mechanical stress transmission performance concurrently and become the urgent needs of researcheres, and the successful research and development of this product will produce huge scientific research and clinical value。
Formhals developed a kind of electrified jet by high-pressure electrostatic field excitation polymer first in 1934, jet is made to solidify the nanofiber obtaining hyperfine structure, the nanofiber prepared by the method possesses superfine fibre diameter, the unique advantage such as large specific surface area and 3-D solid structure, this makes it be increasingly becoming skin wound and repairs the study hotspot in field, its reason is in that: first, tissue engineering bracket constructed by electrostatic spinning technique structurally has analog cell epimatrix (extracellularmatrix, ECM) effect, the adhesion of fibroblast and keratinocyte can be promoted, propagation and migration；Secondly, in the electrostatic spinning solution of the present invention with there is good biocompatibility collagen protein for raw material, this nano fiber scaffold can substantially speed up wound healing。3rd, alginic acid dressing is a class Wound dressing medicine of current comparative maturity, and when namely alginate is by contacting with wound exudate, the exchange of generation ion is thus being formed and having gelatinous alginic acid dressing。Moist environment needed for wound healing and the advantage such as wound surface stickiness is good is created owing to this dressing has, become a class bio-medical material of good performance (alginate dressing application present situation and progress, China's Reconstructive surgery magazine, 2014,28 (2))。The present invention on this basis, with the natural high molecular substance such as alginic acid and chitosan for raw material, at Ca2+Ion exchange under formed there is the compound porous gel of micro-nano structure, can not only play the barrier action being isolated from the outside, and can also induced platelet activation, promote wound surface hemostasis and work in coordination with bacteriostasis。
Although there being Many researchers to develop multiple Medical wounded surface dressing at present, at present any prepare nano fibrous membrane with porous gel as VAC dressing the pertinent literature being applied to wound repair field by pure natural high polymer, the preparation method of the application number nanometer fiber immobilization beta-D-galactosidase that has been the patent report of " CN201010190222.6 ", but carry out the correlational study of biomedical sector。The preparation method that the patent that application number is " CN103705970A " with " CN101224310B " reports a kind of fibroin base biology wound dressing and a kind of medical wound dressing being loaded with antibacterials nanoparticle respectively, but there is no the correlational study of oxygen permeability and the micro-nano structure combine dressing significantly improving Medical wounded surface dressing。
Summary of the invention
It is an object of the invention to solve the problems referred to above, a kind of porous pluralgel-nanofiber oxygen flow dressing with excellent oxygen permeability, bacteriostasis and short more function and preparation method thereof is provided, this oxygen flow dressing combines the features such as appearance structure controlled, gel rapid shaping, electrostatic spinning film forming, there is excellent oxygen permeability, water absorption, bacteriostasis and short more effect, wound surface physiology moist environment can be kept, promote wound healing and hemostasis, wound infection can be prevented and accelerate recovery from illness, it is adaptable to the multiple wound surface such as wound, burn, ulcer。
For achieving the above object, the present invention adopts the following technical scheme that and is achieved:
A kind of porous pluralgel-nanofiber oxygen flow dressing, including the nano fibrous membrane of the pluralgel on upper strata and lower floor；Pluralgel and nano fibrous membrane are all loaded with antibacterials；Pluralgel is sponge state when dry, is gel state after moisture absorption；Pluralgel is made up of the chitosan that mass ratio is 1:3 and sodium alginate, and nano fibrous membrane is made up of collagen protein。
The sponge porosity 50～85% of described upper strata pluralgel, the diameter of lower floor's nano fibrous membrane is 200～600nm；The dressing oxygen transmission rate prepared is 50～60%, and water absorption rate is 1000～2500%。
The preparation method of a kind of porous pluralgel-nanofiber oxygen flow dressing, comprises the following steps:
1) collagen protein is placed in the acetum that mass fraction is 0.5～2mol/L, is configured to the collagen solution that mass concentration is 4～12%；
2) by step 1) in obtain collagen solution prepare into collagen protein nano fibrous membrane by electrostatic spinning；
3) by step 2) in prepare nano fibrous membrane be dried process to remove residual solvent；Cross-link in the cross-linking agent steam that volumetric concentration is 0.5～5% subsequently；
4) dissolving the chitosan in mass concentration is be configured to chitosan solution in 2% acetic acid, then calcium chloride water it is added thereto to, then in 25～60 DEG C of water-baths, continue stirring 5h, then pH value is regulated to 5, and stir to being completely dissolved under room temperature, stand overnight, obtain the mixed solution of chitosan and calcium chloride；Wherein, in mixed solution, the mass ratio of calcium chloride and chitosan is 1:2～2:1；
5) mixed solution is directly instilled in sodium alginate soln, obtain the three-decker containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate, and inside is the microcapsule gel of liquid；Being taken out by microcapsule gel and rinsing surface to pH value is 6, and dried to humidity is 15%；Then be added thereto to account for the plasticizer of microcapsule gel quality 0.1～0.5% then,；Wherein, in mixed solution, the mass ratio of the gross mass of chitosan and calcium chloride and sodium alginate is 1:5～5:1；
6) by step 3) in the nanofiber for preparing be placed in 10 × 10cm2Mould in, by step 5) in the microcapsule gel being added with plasticizer that obtains be placed in this mould and spread out, prepare forming double-layer composite by lyophilization or vacuum drying method；Fiber and chitosan/alginic acid gel mass ratio are 1:40～1:20, and drying time is 6～120h。
Described step 2) in, concrete electrostatic spinning is by step 1) in obtain collagen solution be fed in 10mL syringe, coutroi velocity is 0.5～2mL/h, the voltage of electrostatic spinning is 10～35kV, receiving range is 5～20cm, the time of Electrospun is 6～96h, the temperature of electrostatic spinning and relative humidity respectively 5～35 DEG C and 20～80%。
Described step 3) in, dried is to carry out in the vacuum drying oven that temperature is 25～35 DEG C, and drying time is 1～48h。
Described step 3) in, cross-linking agent adopts glutaraldehyde or oxirane, and the time of cross-linking reaction is 4～24h。
Described step 4) in, the mass concentration of chitosan solution is 0.1～10%, contains the calcium chloride of 0.5～5mg in every milliliter of calcium chloride water。
Described step 5) in, distilled water flushing surface is used in microcapsule gel taking-up；Dried is to use filter paper wipe dry, then air-dry。
Described step 5) in, the mass concentration of sodium alginate aqueous solution is 1～5%, and plasticizer adopts glycerol, Polyethylene Glycol, sorbitol or ethylene glycol。
Described step 5) in, the response time of mixed solution and sodium alginate soln is 4～24h, and reaction temperature is 25～40 DEG C。
Compared with prior art, the method have the advantages that
Porous pluralgel of the present invention-nanofiber oxygen flow dressing, lower floor is the Electrospun nano-fibers contacted with wound surface, and it can absorb transudate the load antibacterial medicines of wound surface；Upper strata is by natural safety non-toxic and has the porous pluralgel that the chitosan of biological degradability is cross-linked to form with alginic acid, its mechanical property can meet the instructions for use of dressing, and there is excellent oxygen permeability, moisture-inhibiting and every bacterium function, to reaching to shorten wound healing time, the purpose of wound infection rate is greatly lowered。
The preparation method of porous pluralgel of the present invention-nanofiber oxygen flow dressing, cost of material is low, human body and environment are not resulted in adverse effect, whole technical process is simple to operate, it is prone to large-scale production, porous pluralgel-nanofiber the combine dressing of gained not only has fabulous oxygen permeability, antibiotic property and biocompatibility, blood with wound surface place, tissue can form gel after contacting with surrounding skin, there is hemostasis and filming performance, not only can absorb sepage, keep periwound moist environment, and can long lasting for release antibacterials, thus preventing wound infection。Also good application is had in wound repair field。
The present invention utilizes electrostatic spinning technique to prepare collagen protein nano fibrous membrane, subsequently the mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, adopts freeze-drying to prepare fiber-chitosan/alginic acid gel pluralgel two-layer compound thing subsequently。The invention provides a kind of new bio Wound dressing and preparation method thereof, preparation technology is simple, mild condition, the biological wound-surface dressing oxygen permeability obtained is excellent, oxygen transmission rate is up to 50-60%, the combine dressing of preparation has good oxygen permeability and anti-microbial property, it is possible to improves the efficiency of wound healing and prevents wound infection, can remarkably promote wound healing simultaneously。Therefore, the modified micro-nano structure composite that prepared by the present invention can be applied to biomedical sector as a kind of good VAC dressing materials。
Accompanying drawing explanation
Fig. 1 is the structural representation of porous of the present invention pluralgel-nanofiber oxygen flow dressing；
Fig. 2 is the micro-structure diagram of porous of the present invention pluralgel-nanofiber oxygen flow dressing, and wherein Fig. 2-1 is the microstructure schematic diagram of upper strata pluralgel, and Fig. 2-2 is the microstructure schematic diagram of lower floor's nano fibrous membrane；
Fig. 3 is the oxygen permeability test result figure of porous of the present invention pluralgel-nanofiber oxygen flow dressing；
Fig. 4 is the bacteriostasis property result of the test figure of porous of the present invention pluralgel-nanofiber oxygen flow dressing；Wherein, Fig. 4-1 is escherichia coli fungistatic effect figure, Fig. 4-2 is staphylococcus aureus fungistatic effect figure；
Fig. 5 is the vivo degradation HE coloration result figure of porous of the present invention pluralgel-nanofiber oxygen flow dressing；Wherein, a is collagen protein nano fibrous membrane, and b is chitosan/sodium alginate compound pluralgel, c porous pluralgel-nanofiber oxygen flow dressing；
Fig. 6 is the wound healing result of the test of different dressing, and wherein, A is collagen protein nano fibrous membrane, and B is chitosan/sodium alginate compound pluralgel, C porous pluralgel-nanofiber oxygen flow dressing。
Detailed description of the invention
Below by specific embodiment, technical scheme is described further, its object is to help and be better understood from present disclosure, but the protection domain that these specific embodiments do not limit the present invention in any way。
Referring to Fig. 1, porous pluralgel of the present invention-nanofiber oxygen flow dressing, including the nano fibrous membrane 2 of the pluralgel 1 on upper strata and lower floor；Pluralgel and nano fibrous membrane are all loaded with antibacterials；As in figure 2 it is shown, pluralgel is sponge state when dry, moisture absorption rear guard's gel state；Pluralgel is made up of the chitosan that mass ratio is 1:3 and sodium alginate, and nano fibrous membrane is made up of collagen protein。The sponge porosity 50～85% of upper strata pluralgel, the diameter of lower floor's nano fibrous membrane is 200～600nm；The dressing oxygen transmission rate prepared is 50～60%, and water absorption rate is 1000～2500%。
Embodiment 1
(1) collagen protein is placed in the acetum that mass fraction is 1mol/L, is configured to the collagen solution that mass concentration is 8% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1mL/h, and the voltage of electrostatic spinning is 30kV, and receiving range is 15cm, and the time of Electrospun is 24h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 25 DEG C and 45%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 30 DEG C, dries 6h to remove residual solvent；Cross-linking in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, 1mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 60 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:2。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:3, adding weight ratio in the solution prepared is that the glycerol of 0.1% is as plasticizer, response time is 12h, reaction temperature is 25 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by freeze-drying；Fiber and chitosan/alginic acid gel mass ratio are 1:30, and freeze-drying time is 48h。
Embodiment 2
(1) collagen protein is placed in the acetum that mass fraction is 1mol/L, is configured to the collagen solution that mass concentration is 8% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1mL/h, and the voltage of electrostatic spinning is 30kV, and receiving range is 15cm, and the time of Electrospun is 48h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 25 DEG C and 45%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 30 DEG C, dries 12h to remove residual solvent；Cross-linking in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 2%, and crosslinking time is 12h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, 1mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 60 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:1。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:2, adding weight ratio in the solution prepared is that the glycerol of 0.2% is as plasticizer, response time is 24h, reaction temperature is 25 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by freeze-drying；Fiber and chitosan/alginic acid gel mass ratio are 1:30, and freeze-drying time is 48h。
Embodiment 3
(1) collagen protein is placed in the acetum that mass fraction is 1mol/L, is configured to the collagen solution that mass concentration is 8% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1mL/h, and the voltage of electrostatic spinning is 30kV, and receiving range is 15cm, and the time of Electrospun is 24h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 25 DEG C and 45%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 30 DEG C, dries 6h to remove residual solvent；Cross-linking in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, 1mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 60 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:2。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:1, adding weight ratio in the solution prepared is that the glycerol of 0.5% is as plasticizer, response time is 20h, reaction temperature is 25 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by freeze-drying；Fiber and chitosan/alginic acid gel mass ratio are 1:35, and freeze-drying time is 72h。
Embodiment 4
(1) collagen protein is placed in the acetum that mass fraction is 1mol/L, is configured to the collagen solution that mass concentration is 8% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1mL/h, and the voltage of electrostatic spinning is 30kV, and receiving range is 15cm, and the time of Electrospun is 24h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 25 DEG C and 45%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 30 DEG C, dries 6h to remove residual solvent；Cross-linking in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 4% in 2% glacial acetic acid aqueous solution, 1mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 60 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:2。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 4% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:2, adding weight ratio in the solution prepared is that the glycerol of 0.2% is as plasticizer, response time is 24h, reaction temperature is 30 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by freeze-drying；Fiber and chitosan/alginic acid gel mass ratio are 1:35, and freeze-drying time is 72h。
Embodiment 5
(1) collagen protein is placed in the acetum that mass fraction is 1mol/L, is configured to the collagen solution that mass concentration is 8% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1mL/h, and the voltage of electrostatic spinning is 30kV, and receiving range is 15cm, and the time of Electrospun is 24h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 25 DEG C and 45%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 30 DEG C, dries 6h to remove residual solvent；Cross-linking in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 3% in 2% glacial acetic acid aqueous solution, 1mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 60 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:1。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 3% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:2, adding weight ratio in the solution prepared is that the glycerol of 0.1% is as plasticizer, response time is 24h, reaction temperature is 25 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by freeze-drying；Fiber and chitosan/alginic acid gel mass ratio are 1:30, and freeze-drying time is 48h。
Embodiment 6
(1) collagen protein is placed in the acetum that mass fraction is 1mol/L, is configured to the collagen solution that mass concentration is 8% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1mL/h, and the voltage of electrostatic spinning is 30kV, and receiving range is 15cm, and the time of Electrospun is 24h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 25 DEG C and 45%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 30 DEG C, dries 6h to remove residual solvent；Cross-linking in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, 1mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 60 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:2。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:3, adding weight ratio in the solution prepared is that the glycerol of 0.2% is as plasticizer, response time is 12h, reaction temperature is 25 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by freeze-drying；Fiber and chitosan/alginic acid gel mass ratio are 1:40, and freeze-drying time is 96h。
Embodiment 7
(1) collagen protein is placed in the acetum that mass fraction is 0.5mol/L, is configured to the collagen solution that mass concentration is 4% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 0.5mL/h, and the voltage of electrostatic spinning is 10kV, and receiving range is 5cm, and the time of Electrospun is 6h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 5 DEG C and 20%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 25 DEG C, dries 1h to remove residual solvent；Cross-linking in ethylene oxide vapor subsequently, the volumetric concentration of oxirane is 0.5%, and crosslinking time is 4h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 0.1% in 2% glacial acetic acid aqueous solution, 0.5mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 25 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 2:1。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 1% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 5:1, adding weight ratio in the solution prepared is that the Polyethylene Glycol of 0.3% is as plasticizer, response time is 4h, reaction temperature is 30 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by boulton process；Fiber and chitosan/alginic acid gel mass ratio are 1:25, and the vacuum drying time is 6h。
Embodiment 8
(1) collagen protein is placed in the acetum that mass fraction is 1.5mol/L, is configured to the collagen solution that mass concentration is 9% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 1.5mL/h, and the voltage of electrostatic spinning is 20kV, and receiving range is 10cm, and the time of Electrospun is 72h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 15 DEG C and 40%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 28 DEG C, dries 30h to remove residual solvent；Cross-linking in ethylene oxide vapor subsequently, the volumetric concentration of oxirane is 3%, and crosslinking time is 18h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 7% in 2% glacial acetic acid aqueous solution, 3mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 35 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 2:3。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 3% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:4, adding weight ratio in the solution prepared is that the sorbitol of 0.4% is as plasticizer, response time is 10h, reaction temperature is 35 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by boulton process；Fiber and chitosan/alginic acid gel mass ratio are 1:25, and the vacuum drying time is 36h。
Embodiment 9
(1) collagen protein is placed in the acetum that mass fraction is 2mol/L, is configured to the collagen solution that mass concentration is 12% (w/w)；
(2) being fed in 10mL syringe by the collagen solution obtained in step (1), coutroi velocity is 2mL/h, and the voltage of electrostatic spinning is 35kV, and receiving range is 20cm, and the time of Electrospun is 96h。Electrostatic spinning is adopted to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity respectively 35 DEG C and 80%；
(3) nano fibrous membrane prepared in step (2) is placed in vacuum drying oven, and at 35 DEG C, dries 48h to remove residual solvent；Cross-linking in ethylene oxide vapor subsequently, the volumetric concentration of oxirane is 5%, and crosslinking time is 24h；
(4) dissolving the chitosan in mass concentration is be configured to the chitosan solution that mass concentration is 10% in 2% glacial acetic acid aqueous solution, 5mg/mL calcium chloride water by preparation, after ultrasonic 5min, it is slowly added to the above-mentioned chitosan solution prepared, 55 DEG C of water-baths continue stirring 5h, wherein CaCl2With the mass percentage concentration of CS than for 1:1。Regulate pH to 5, stir under room temperature to being completely dissolved, stand overnight。It is the sodium alginate soln of 5% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, removes precipitation。
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction in sodium alginate soln and forms microcapsule, finally give containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate three layers altogether, inside is the gel of the microcapsule of liquid, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dry it is about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:5, adding weight ratio in the solution prepared is that the ethylene glycol of 0.5% is as plasticizer, response time is 15h, reaction temperature is 40 DEG C。
(6) nanofiber prepared in step (3) is placed in 10 × 10cm2In molding jig, the gel of the microcapsule being added with glycerol obtained in step (5) is placed in this mould and spreads out, prepare forming double-layer composite by boulton process；Fiber and chitosan/alginic acid gel mass ratio are 1:20, and the vacuum drying time is 120h。
Referring to Fig. 3 to Fig. 6, the effect of the present invention is as follows:
As shown in Figure 3, the oxygen permeability test result of porous pluralgel of the present invention-nanofiber oxygen flow dressing shows: gel sponge has higher oxygen transmission rate than nanofiber, and porous of the present invention pluralgel-nanofiber oxygen flow dressing has better oxygen permeability than two kinds of independent components。
As shown in Figure 4, porous pluralgel of the present invention-nanofiber oxygen flow dressing is respectively provided with significant inhibition for escherichia coli (gram negative bacteria) and staphylococcus aureus (gram positive bacteria)。Wherein, Fig. 4-1 reflects the nanofiber of preparation, sponge and combine dressing to colibacillary fungistatic effect (showing with antibacterial circle diameter)；Fig. 4-2 reflects these three material to staphylococcus aureus fungistatic effect (showing with antibacterial circle diameter)。
As shown in the HE coloration result of Fig. 5, porous pluralgel of the present invention-nanofiber oxygen flow dressing has excellent vivo degradation performance, and has a large amount of new vessels to generate after material is implanted。Wherein, a is collagen protein nano fibrous membrane, and b is chitosan/sodium alginate compound pluralgel, c porous pluralgel-nanofiber oxygen flow dressing；Experimental result confirms: porous pluralgel of the present invention-nanofiber oxygen flow dressing has better vivo degradation performance than simple gel sponge and nanofiber within the same implantation cycle。
As shown in Figure 6, different dressing obtain effect for wound healing and there is notable difference, and wherein, A is collagen protein nano fibrous membrane, and B is chitosan/sodium alginate compound pluralgel, C porous pluralgel-nanofiber oxygen flow dressing。Result shows: after the wound healing that the porous pluralgel prepared through the present invention-nanofiber oxygen flow dressing processes, re-epithelialization degree is higher, illustrates that this combine dressing is best for the facilitation effect of wound healing。
What Formulas I represented respectively is the chemical structural formula of chitosan and alginic acid, represents the structural formula forming gel simultaneously, forms gel under chemical cross-linking agent calcium ion effect；Wherein, Formulas I is as follows:
Above-described embodiment set forth in detail the preferred embodiment of the present invention, and the present invention is made that detailed description, not limits the present invention with above-described embodiment。Skilled person realizes that when without departing from the technical characteristic given by technical solution of the present invention and scope increase that technical characteristic is made or the replacement with some same contents all should belong to protection scope of the present invention。
Claims (9)
1. porous pluralgel-nanofiber oxygen flow dressing, it is characterised in that: include the pluralgel (1) on upper strata and the nano fibrous membrane (2) of lower floor；Pluralgel and nano fibrous membrane are all loaded with antibacterials；Pluralgel is sponge state when dry, is gel state after moisture absorption；Pluralgel is made up of the chitosan that mass ratio is 1:3 and sodium alginate, and nano fibrous membrane is made up of collagen protein；
The sponge porosity 50～85% of described upper strata pluralgel, the diameter of lower floor's nano fibrous membrane is 200～600nm；The dressing oxygen transmission rate prepared is 50～60%, and water absorption rate is 1000～2500%。
2. the preparation method of porous pluralgel-nanofiber oxygen flow dressing, it is characterised in that comprise the following steps:
1) collagen protein is placed in the acetum that mass fraction is 0.5～2mol/L, is configured to the collagen solution that mass concentration is 4～12%；
2) by step 1) in obtain collagen solution prepare into collagen protein nano fibrous membrane by electrostatic spinning；
3) by step 2) in prepare nano fibrous membrane be dried process to remove residual solvent；Cross-link in the cross-linking agent steam that volumetric concentration is 0.5～5% subsequently；
4) dissolving the chitosan in mass concentration is be configured to chitosan solution in 2% acetic acid, then calcium chloride water it is added thereto to, then in 25～60 DEG C of water-baths, continue stirring 5h, then pH value is regulated to 5, and stir to being completely dissolved under room temperature, stand overnight, obtain the mixed solution of chitosan and calcium chloride；Wherein, in mixed solution, the mass ratio of calcium chloride and chitosan is 1:2～2:1；
5) mixed solution is directly instilled in sodium alginate soln, obtain the three-decker containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate, and inside is the microcapsule gel of liquid；Being taken out by microcapsule gel and rinsing surface to pH value is 6, and dried to humidity is 15%；Then be added thereto to account for the plasticizer of microcapsule gel quality 0.1～0.5% then,；Wherein, in mixed solution, the mass ratio of the gross mass of chitosan and calcium chloride and sodium alginate is 1:5～5:1；
6) by step 3) in the nanofiber for preparing be placed in 10 × 10cm2Mould in, by step 5) in the microcapsule gel being added with plasticizer that obtains be placed in this mould and spread out, prepare forming double-layer composite by lyophilization or vacuum drying method；Fiber and chitosan/alginic acid gel mass ratio are 1:40～1:20, and drying time is 6～120h。
3. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2, it is characterized in that: described step 2) in, concrete electrostatic spinning is by step 1) in obtain collagen solution be fed in 10mL syringe, coutroi velocity is 0.5～2mL/h, the voltage of electrostatic spinning is 10～35kV, receiving range is 5～20cm, and the time of Electrospun is 6～96h, the temperature of electrostatic spinning and relative humidity respectively 5～35 DEG C and 20～80%。
4. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2, it is characterised in that: described step 3) in, dried is to carry out in the vacuum drying oven that temperature is 25～35 DEG C, and drying time is 1～48h。
5. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2 or 4, it is characterised in that: described step 3) in, cross-linking agent adopts glutaraldehyde or oxirane, and the time of cross-linking reaction is 4～24h。
6. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2, it is characterised in that: described step 4) in, the mass concentration of chitosan solution is 0.1～10%, contains the calcium chloride of 0.5～5mg in every milliliter of calcium chloride water。
7. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2, it is characterised in that: described step 5) in, distilled water flushing surface is used in microcapsule gel taking-up；Dried is to use filter paper suck dry moisture, then air-dry。
8. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2, it is characterized in that: described step 5) in, the mass concentration of sodium alginate aqueous solution is 1～5%, and plasticizer adopts glycerol, Polyethylene Glycol, sorbitol or ethylene glycol。
9. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 2 or 7 or 8, it is characterised in that: described step 5) in, the response time of mixed solution and sodium alginate soln is 4～24h, and reaction temperature is 25～40 DEG C。
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