CN104491914A - Porous complex gel-nanofiber oxygen permeation dressing and preparation method thereof - Google Patents
Porous complex gel-nanofiber oxygen permeation dressing and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a porous complex gel-nanofiber oxygen permeation dressing and a preparation method thereof. The method comprises the following steps: obtaining a collagen nanofiber membrane by adopting a collagen solution by virtue of an electrostatic spinning method; performing chemical crosslinking on two natural and non-toxic macromolecule polysaccharides such as chitosan and alginic acid, and preparing complex gel; and preparing the porous sponge-nanofiber double-layer composite material in a freeze-drying mode, wherein the upper gel layer of the dressing is in a sponge state during drying and is in a gel state after moisture absorption. The biological wound dressing is prepared under the condition that any toxic chemical crosslinking agent is not added, the process is simple, and the safety is high. The prepared biological wound dressing disclosed by the invention has the characteristics of excellent oxygen permeation performance, water-absorbing quality, water permeability, moisture penetrability and high biocompatibility, a physiological wet environment of the wound surface can be maintained, wound healing and effective haemostasis are promoted, wound infection can be avoided, the recovery is accelerated, the wound surface can be effectively recovered, and the dressing is applied to multiple wound surfaces such as trauma, burns and ulceration.
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 a kind of porous pluralgel-nanofiber oxygen flow dressing and preparation method thereof.
Background technology
Closed negative pressure drainage (Vacuum-assisted closure, VAC) technology is as a kind of new technique 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 from 1998 and achieve a collection of original result of study, we find: application VAC treatment can make wound surface environment continue to be in hypoxia, there are some researches show, although in wound healing early process, anaerobic environment is conducive to wound surface angeogenesis, but along with the carrying out of wound healing, increasing organizes oxygen supply that local cellular proliferation can be made to 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 accelerate wound healing (oxygen, ozone and negative pressure drainage use in conjunction are to the therapeutical effect of wound surface, J Trauma Surg, 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 may be relevant with the microcirculation disturbance and bacteria growing inhibiting improving wound tissue.
The dressing used for VAC technology is at present studied relatively less, 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 the drawback that healing stage is longer, wound cicatrix obvious, the process of changing dressings causes patient suffering, the environment that gauze can cause wound surface drier, wound surface is dewatered, causes incrustation.And the epithelization of the incrustation of wound surface to wound surface has obvious obstacle (development of collagen/chitosan composite table skin growth factor thrombin sponge dressing, ACAD J GCP, 2004,20 (5)).Usual hydrogel wound dressing has absorption wound fluid, wound surface is kept to clean moistening, reduce cicatrization, the advantages such as wound healing, but not there is antibiotic property, preventing existing defects in wound infection, while this often just requires that 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.) be main, VAC technology can form a kind of shearing force at medical foam and wound interface, this mechanical stress is considered to the effect promoting granulation tissue growth and angiogenesis.But, current existing medical plural gel dressing cannot realize the conduction of this stress preferably, therefore exploitation has the urgent needs that the Medical dressing for skin wound that excellent oxygen permeability, bacteriostasis property and mechanical stress transmit performance has become researcheres concurrently, 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 comparatively unique advantage such as Large ratio surface sum 3-D solid structure, this makes it become the study hotspot in skin wound reparation field gradually, its reason is: first, tissue engineering bracket constructed by electrostatic spinning technique structurally has analog cell epimatrix (extracellular matrix, ECM) effect, the adhesion of fibroblast and keratinocyte can be promoted, propagation and migration, secondly, to have the collagen protein of good biocompatibility for raw material in electrostatic spinning solution of the present invention, this nano fiber scaffold significantly can accelerate wound healing.Three, alginic acid dressing is a class Wound dressing medicine of current comparative maturity, when namely alginate is by contacting with wound exudate, ion exchange occurs thus formed to have gelatinous alginic acid dressing.Due to this dressing have create moist environment needed for wound healing, with the advantage such as wound surface stickiness is good, become 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 Ca
2+ion exchange under formed there is the compound porous gel of micro-nano structure, the barrier action be isolated from the outside can not only be played, and can also induced platelet activation, promote wound surface hemostasis and work in coordination with bacteriostasis.
Although the multiple Medical wounded surface dressing that had Many researchers to develop at present, at present anyly prepare nano fibrous membrane and porous gel as VAC dressing by pure natural high polymer and be applied to the pertinent literature in wound repair field, the preparation method that patent reports nanometer fiber immobilization beta-D-galactosidase that application number is " CN201010190222.6 ", but do not carry out the correlational study of biomedical sector.The patent that application number is " CN103705970A " and " CN101224310B " reports the biological wound dressing of a kind of fibroin base and a kind of preparation method being loaded with the medical wound dressing of 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
The object of the invention is to solve the problem, there is provided a kind of and there is porous pluralgel-nanofiber oxygen flow dressing of excellent oxygen permeability, bacteriostasis and short more function and preparation method thereof, this oxygen flow dressing combines the features such as appearance structure is 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, can wound infection be prevented and accelerate recovery from illness, being applicable to the multiple wound surface such as wound, burn, ulcer.
For achieving the above object, the present invention adopts following technical scheme to be achieved:
A kind of porous pluralgel-nanofiber oxygen flow dressing, comprises the pluralgel on upper strata and the nano fibrous membrane of lower floor; On pluralgel and nano fibrous membrane, all load has antibacterials; Pluralgel is sponge state when drying, is gel state after moisture absorption; Pluralgel is that the chitosan of 1:3 and sodium alginate are made by mass ratio, 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; Obtained dressing oxygen transmission rate is 50 ~ 60%, and water absorption rate is 1000 ~ 2500%.
A preparation method for 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 mixed with the collagen solution that mass concentration is 4 ~ 12%;
2) by step 1) in obtain collagen solution be prepared into collagen protein nano fibrous membrane by electrostatic spinning;
3) by step 2) in obtained nano fibrous membrane carry out dried to remove residual solvent; Be be cross-linked in the cross-linking agent steam of 0.5 ~ 5% subsequently in volumetric concentration;
4) chitosan being dissolved in mass concentration is be mixed with chitosan solution in 2% acetic acid, then calcium chloride water is added wherein, then continue to stir 5h in 25 ~ 60 DEG C of water-baths, then adjust ph to 5, and in stirred at ambient temperature to dissolving completely, hold over night, obtains 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 liquid microcapsule gel; Being taken out by microcapsule gel and rinsing surface to pH value is 6, and dried to humidity is 15%; Then, more wherein add the plasticizer accounting for microcapsule gel quality 0.1 ~ 0.5%; Wherein, in mixed solution, the gross mass of chitosan and calcium chloride and the mass ratio of sodium alginate are 1:5 ~ 5:1;
6) by step 3) in the nanofiber for preparing be placed in 10 × 10cm
2mould in, by step 5) in the microcapsule gel being added with plasticizer that obtains be placed in this mould and spread out, obtain 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, and the temperature of electrostatic spinning and relative humidity are respectively 5 ~ 35 DEG C and 20 ~ 80%.
Described step 3) in, dried is carry out in the vacuum drying oven of 25 ~ 35 DEG C in temperature, 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%, the calcium chloride containing 0.5 ~ 5mg in every milliliter of calcium chloride water.
Described step 5) in, by microcapsule gel taking-up distilled water flushing surface; Dried uses 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 present invention has following beneficial effect:
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 the transudate of wound surface and load antibacterial medicines; Upper strata is by natural safety non-toxic and has the porous pluralgel that the chitosan of biological degradability and alginic acid be cross-linked to form, 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 shortening wound healing time, significantly reduce the object of wound infection rate.
The preparation method of porous pluralgel of the present invention-nanofiber oxygen flow dressing, cost of material is low, adverse effect can not be caused to human body and environment, whole technical process is simple to operate, be easy to large-scale production, porous pluralgel-nanofiber the combine dressing of gained not only has fabulous oxygen permeability, antibiotic property and biocompatibility, with the blood at wound surface place, gel can be formed after tissue contacts with surrounding skin, there is hemostasis and filming performance, not only Absorbable rod sepage, keep periwound moist environment, and can sustained release antibacterials for a long time, thus prevent 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 formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, 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, can improve the efficiency of wound healing and prevent wound infection, simultaneously can remarkable wound healing.Therefore, the modification 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 of the present invention is described further, its object is to help better to understand content of the present invention, but the protection domain that these specific embodiments do not limit the present invention in any way.
See Fig. 1, porous pluralgel of the present invention-nanofiber oxygen flow dressing, comprises the pluralgel 1 on upper strata and the nano fibrous membrane 2 of lower floor; On pluralgel and nano fibrous membrane, all load has antibacterials; As shown in Figure 2, pluralgel is sponge state when drying, moisture absorption rear guard gel state; Pluralgel is that the chitosan of 1:3 and sodium alginate are made by mass ratio, 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; Obtained dressing oxygen transmission rate 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 mixed with the collagen solution that mass concentration is 8% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 25 DEG C and 45%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 30 DEG C dry 6h to remove residual solvent; Crosslinked in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, by the 1mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 60 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:2 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:3, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 8% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 25 DEG C and 45%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 30 DEG C dry 12h to remove residual solvent; Crosslinked in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 2%, and crosslinking time is 12h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, by the 1mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 60 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:1 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:2, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 8% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 25 DEG C and 45%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 30 DEG C dry 6h to remove residual solvent; Crosslinked in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, by the 1mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 60 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:2 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:1, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 8% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 25 DEG C and 45%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 30 DEG C dry 6h to remove residual solvent; Crosslinked in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 4% in 2% glacial acetic acid aqueous solution, by the 1mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 60 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:2 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 4% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:2, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 8% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 25 DEG C and 45%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 30 DEG C dry 6h to remove residual solvent; Crosslinked in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 3% in 2% glacial acetic acid aqueous solution, by the 1mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 60 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:1 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 3% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:2, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 8% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 25 DEG C and 45%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 30 DEG C dry 6h to remove residual solvent; Crosslinked in glutaraldehyde steam subsequently, the volumetric concentration of glutaraldehyde is 1%, and crosslinking time is 6h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 2% in 2% glacial acetic acid aqueous solution, by the 1mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 60 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:2 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 2% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:3, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 4% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 5 DEG C and 20%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 25 DEG C dry 1h to remove residual solvent; Crosslinked in ethylene oxide vapor subsequently, the volumetric concentration of oxirane is 0.5%, and crosslinking time is 4h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 0.1% in 2% glacial acetic acid aqueous solution, by the 0.5mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 25 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 2:1 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 1% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 5:1, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 9% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 15 DEG C and 40%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 28 DEG C dry 30h to remove residual solvent; Crosslinked in ethylene oxide vapor subsequently, the volumetric concentration of oxirane is 3%, and crosslinking time is 18h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 7% in 2% glacial acetic acid aqueous solution, by the 3mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 35 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 2:3 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 3% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:4, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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 mixed with the collagen solution that mass concentration is 12% (w/w);
(2) be 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.Adopt electrostatic spinning to prepare collagen protein nanofiber, the temperature of electrostatic spinning and relative humidity are respectively 35 DEG C and 80%;
(3) nano fibrous membrane obtained in step (2) is placed in vacuum drying oven, and at 35 DEG C dry 48h to remove residual solvent; Crosslinked in ethylene oxide vapor subsequently, the volumetric concentration of oxirane is 5%, and crosslinking time is 24h;
(4) chitosan being dissolved in mass concentration is be mixed with the chitosan solution that mass concentration is 10% in 2% glacial acetic acid aqueous solution, by the 5mg/mL calcium chloride water of preparation, after ultrasonic 5min, slowly be added in the above-mentioned chitosan solution prepared, continue in 55 DEG C of water-baths to stir 5h, wherein CaCl
2compare for 1:1 with the mass percentage concentration of CS.Regulate pH to 5, stirred at ambient temperature extremely dissolves completely, hold over night.It is the sodium alginate soln of 5% that alginic acid dissolves preparation mass percentage concentration at ultra-pure water, centrifugal after stirring, and removes precipitation.
(5) mixed solution of chitosan and calcium chloride is directly instilled reaction formation microcapsule in sodium alginate soln, finally obtain containing chitosan precipitation layer, chitosan/sodium alginate complexation layer and calcium alginate coacervate totally three layers, inside is the gel of liquid microcapsule, taking-up distilled water flushing surface is about 6 to pH value, use filter paper wipe dry, air-dryly be about 15% to humidity, wherein chitosan/calcium chloride and sodium alginate mass ratio are 1:5, in prepared solution, add weight ratio 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 × 10cm
2in molding jig, the gel being added with the microcapsule of glycerol obtained in step (5) is placed in this mould and spreads out, obtain 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.
See Fig. 3 to Fig. 6, effect of the present invention is as follows:
As shown in Figure 3, the oxygen permeability test result display of porous pluralgel of the present invention-nanofiber oxygen flow dressing: 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 all has 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 kinds of materials to staphylococcus aureus fungistatic effect (with antibacterial circle diameter display).
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 obtains 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 of the porous pluralgel-nanofiber oxygen flow dressing process prepared through the present invention, re-epithelialization degree is higher, illustrates that this combine dressing is best for the facilitation effect of wound healing.
The chemical structural formula of what formula I represented respectively is chitosan and alginic acid, representative simultaneously forms the structural formula of gel, under the effect of chemical cross-linking agent calcium ion, form gel; Wherein, formula I is as follows:
Above-described embodiment set forth in detail preferred embodiment of the present invention, to invention has been detailed description, not limits the present invention with above-described embodiment.Those skilled in the art should recognize when the technical characteristic do not departed from given by technical solution of the present invention and scope, the increase do technical characteristic or with the replacement of some same contents, all should belong to protection scope of the present invention.
Claims (10)
1. porous pluralgel-nanofiber oxygen flow dressing, is characterized in that: comprise the pluralgel (1) on upper strata and the nano fibrous membrane (2) of lower floor; On pluralgel and nano fibrous membrane, all load has antibacterials; Pluralgel is sponge state when drying, is gel state after moisture absorption; Pluralgel is that the chitosan of 1:3 and sodium alginate are made by mass ratio, and nano fibrous membrane is made up of collagen protein.
2. porous pluralgel according to claim 1-nanofiber oxygen flow dressing, is characterized in that: the sponge porosity 50 ~ 85% of described upper strata pluralgel, the diameter of lower floor's nano fibrous membrane is 200 ~ 600nm; Obtained dressing oxygen transmission rate is 50 ~ 60%, and water absorption rate is 1000 ~ 2500%.
3. a preparation method for porous pluralgel-nanofiber oxygen flow dressing, is characterized in that, comprise the following steps:
1) collagen protein is placed in the acetum that mass fraction is 0.5 ~ 2mol/L, is mixed with the collagen solution that mass concentration is 4 ~ 12%;
2) by step 1) in obtain collagen solution be prepared into collagen protein nano fibrous membrane by electrostatic spinning;
3) by step 2) in obtained nano fibrous membrane carry out dried to remove residual solvent; Be be cross-linked in the cross-linking agent steam of 0.5 ~ 5% subsequently in volumetric concentration;
4) chitosan being dissolved in mass concentration is be mixed with chitosan solution in 2% acetic acid, then calcium chloride water is added wherein, then continue to stir 5h in 25 ~ 60 DEG C of water-baths, then adjust ph to 5, and in stirred at ambient temperature to dissolving completely, hold over night, obtains 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 liquid microcapsule gel; Being taken out by microcapsule gel and rinsing surface to pH value is 6, and dried to humidity is 15%; Then, more wherein add the plasticizer accounting for microcapsule gel quality 0.1 ~ 0.5%; Wherein, in mixed solution, the gross mass of chitosan and calcium chloride and the mass ratio of sodium alginate are 1:5 ~ 5:1;
6) by step 3) in the nanofiber for preparing be placed in 10 × 10cm
2mould in, by step 5) in the microcapsule gel being added with plasticizer that obtains be placed in this mould and spread out, obtain 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.
4. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3, 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, and the temperature of electrostatic spinning and relative humidity are respectively 5 ~ 35 DEG C and 20 ~ 80%.
5. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3, is characterized in that: described step 3) in, dried is carry out in the vacuum drying oven of 25 ~ 35 DEG C in temperature, and drying time is 1 ~ 48h.
6. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3 or 5, is characterized in that: described step 3) in, cross-linking agent adopts glutaraldehyde or oxirane, and the time of cross-linking reaction is 4 ~ 24h.
7. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3, it is characterized in that: described step 4) in, the mass concentration of chitosan solution is 0.1 ~ 10%, the calcium chloride containing 0.5 ~ 5mg in every milliliter of calcium chloride water.
8. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3, is characterized in that: described step 5) in, by microcapsule gel taking-up distilled water flushing surface; Dried uses filter paper wipe dry, then air-dry.
9. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3, 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.
10. the preparation method of porous pluralgel-nanofiber oxygen flow dressing according to claim 3 or 8 or 9, is characterized 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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