CN106421888A - Keratin-chitosan composite medical dressing and preparation method thereof - Google Patents
Keratin-chitosan composite medical dressing and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a keratin-chitosan composite medical dressing and a preparation method thereof; the dressing is prepared from, by weight, 2-8% of keratin, 0.6-1% of sodium carboxymethyl cellulose, 0.3-0.9% of chitosan, 6-8% of glycerol, and the balance of water. The preparation method comprises: adding keratin and sodium carboxymethyl cellulose into water, adding chitosan and glycerol, mixing well, and performing first lyophilizing; soaking sequentially in CaCl2 solution and NaOH solution, and performing second lyophilizing after soaking is complete; soaking in glycerol aqueous solution, and performing third lyophilizing after soaking is complete. The keratin-chitosan composite medical dressing prepared herein has no immunogenicity, is good in hemostatic effect and high in air permeability, and also has the advantages, such as good biocompatibility and the ability to promote cell growth.
Description
Technical field
The present invention relates to a kind of dressing is and in particular to a kind of keratin, chitosan-based composite dressing for medical use and preparation method thereof.
Background technology
In existing technology and material, relevant medical dressing is generally made up of non-woven fabrics, blotting paper, cotton for wadding, leakproof film,
This paper medical dressing liquid absorption is big, not modification after imbibition, does not produce fracture and reakdown phenomenon.If but this medical dressing is used
When baby, scald, burn patient, dressing is easy to and wound adhesion, is not easily stripped, and is not absorbed by tissue.Cause
This, research and establishment is simulated the matrix of body tissue and function and mounting system as the emphasis of scientific research by the applicant, and with weight
Build or recover the final goal that skin barrier is injured Wound treating.Biological dressing is the wound-surface cover being currently taken seriously, reason
The Wound dressing thought should effectively stop bacterium infection first, accelerates wound healing, should have simultaneously good biocompatibility,
Can control and absorb wound exudate, be suitable for gas and vapor transmission, protection cambium, accelerate the characteristics such as wound healing.
Keratin biomaterials have penetrate into beneficial to cell, the three dimensional scaffold structure of adhesion and propagation, in cell adherence and
Propagation aspect has the characteristic being substantially better than other materials, has nerve-inducing simultaneously, also shows that very well in terms of hemostasis
Effect.Although, the applicant studies discovery keratin for many years and has a lot of advantages in itself, and its independent role is repairing effect
Fruit aspect still has very big room for improvement.
Content of the invention
The defect that the present invention is directed to prior art provides a kind of keratin, chitosan-based composite dressing for medical use and its preparation side
Method.
The purpose of the present invention is to be achieved through the following technical solutions：
A kind of keratin, chitosan-based composite dressing for medical use, the quality hundred of the material composition of described dressing and various composition
Point ratio is：Keratin：2%～8%；Sodium carboxymethylcellulose：0.6%～1%；Shitosan：0.3%～0.9%；Glycerine：6%
～8%；Balance of water.
Further, the material composition of described dressing and the mass percent of various composition are：Keratin：4%～
6%；Sodium carboxymethylcellulose：0.8%～0.9%；Shitosan：0.6%～0.7%；Glycerine：7%；Balance of water.
Further, described keratin is hair-keratin, and described hair-keratin is by human hair, nail or animal wool
Send out the keratin of preparation.
Further, described hair-keratin is appointing in granular hair-keratin and the hair-keratin of gelation
Meaning is a kind of.The concentration of the hair-keratin of described gelation is 0.5～5%, preferably 2～3%.
Further, described dressing also includes CaCl2Solution, NaOH solution and glycerine water solution.
A kind of described keratin, the preparation method of chitosan-based composite dressing for medical use, described preparation method is as follows：
S1：Prepare keratin；
S2：Weigh various composition in proportion, then keratin and sodium carboxymethylcellulose are added to the water to obtain first solution；
S3：In first solution described in shitosan and glycerine are added to step S2, carry out after mixing freezing for the first time
It is dried, this first time freeze-drying freeze-drying 2～5 hours preferably at -15～-25 DEG C；
S4：It is sequentially placed into CaCl after the first time freeze-drying of step S32Soak 2～5 hours and NaOH in solution
Soak 2～5 hours in solution, soak and finish and carry out second freeze-drying again, this second freeze-drying preferably -25～-
Freeze-drying 4～8 hours at 35 DEG C；
S5：Soak 2～8h after second freeze-drying of step S4 in glycerine water solution again, soak and finish to enter again
Row third time freeze-drying, this third time freeze-drying freeze-drying 10～20 hours preferably at -45～-55 DEG C, that is, obtain
Keratin, chitosan-based composite dressing for medical use.
Further, the keratin described in step S1 is hair-keratin, and described hair-keratin is granular hair
Send out any one in keratin and the hair-keratin of gelation.
Further, the concentration of the hair-keratin of described gelation is 0.5～5%, preferably 2～3%.
Further, in step S4, described CaCl2The concentration of solution is 0.5～1% (mass volume ratio), described NaOH
The concentration of solution is 1-2% (mass volume ratio)；In step S5, described glycerine water solution is containing 20%～40% volume hundred
Divide the glycerine water solution of ratio.
Further, the first time freeze-drying described in step S3, freeze-drying to water content is 15～25%,
Till preferably 20%.
Second freeze-drying described in step S4, freeze-drying to water content is less than 15%, preferably 6～
Till 14%, till more preferably 10%.
Third time freeze-drying described in step S5, freeze-drying to water content is less than 5%.
Further, the preparation method of described granular hair-keratin is：①：Degreasing and softening：Weigh hair to enter
Row degreasing and softening；②：Decolouring；③：Cleaning, drying：Hair after decolouring is carried out, and is dried；④：The system of particle
Standby：Dried hair is shredded, and adds liquid nitrogen frozen to grind, obtain hair meal；Meal is placed in ball grinder, puts into
Agate bead, adds physiological saline, rotates, ball milling, till particle diameter reaches 50 μm～90 μm, then even for hair-keratin particle
Slurry；⑤：Purify：Physiological saline is added to be centrifuged in homogenate, abandoning supernatant, and remove top layer and the bottom of precipitation, take
The hair-keratin of stage casing even particle size, plus physiological saline mixing continuation centrifugation, repeat aforesaid operations 2～4 times, still finally
So take the hair-keratin of stage casing even particle size, then obtain highly purified granular hair-keratin, standby after sterilizing.
Further, step 2. in decolorization be：Hair after softening is positioned in de-inking solution and is decoloured,
The composition of described de-inking solution be sodium pyrophosphate 3～5 weight portion, potassium peroxydisulfate 1～2 weight portion, hydrogen peroxide 70～90 parts by volume,
Concentrated ammonia liquor 22～33 parts by volume and water 350～450 parts by volume.
Further, the preparation method of the hair-keratin of described gelation is：Take step 5. in prepared graininess
Hair-keratin, add water, fully mix, be positioned over incubation in incubator, to assuming more homogeneous color and proterties, obtain
To the hair-keratin of gelation, standby after sterilizing.
Further, the hair-keratin of prepared granular hair-keratin and gelation is intended to carry out at sterilizing
Reason；Described sterilization treatment is to adopt cobalt60Irradiation is sterilized.
Further, step detailed process 4. is：Dried hair is shredded, and adds liquid nitrogen frozen to grind, obtain
The hair meal being 0.1～2mm to length；Hair meal is placed in ball grinder, puts into agate bead, add physiological saline, hair
The mass volume ratio sending out meal with physiological saline is 2:5, ball milling 1～3 hour, examine under a microscope particle size, until shape
Become about 60 μm -80 μm of particle diameter；The quantity of described agate bead is：The corresponding diameter 1cm agate bead of the hair meal of every 1g 10 and straight
Footpath 6mm agate bead 50.
Further, the hair-keratin of described gelation can also be prepared by the following method：
(1) hair-keratin becomes salt：Weigh hair, add the mixed solution of second alcohol and water, heating, backflow, in backflow
Dropping aqueous slkali, is to terminate when 6.5～7.5 to pH value, then proceedes to stir, make reaction completely, be filtrated to get hair-keratin
Salt, dry, pulverize to micron order, is lyophilized, that is, obtains hair-keratin sulfonic acid salt powder；
(2) preparation of hair-keratin gel：Weigh hair-keratin sulfonic acid salt powder, add water, fully mix, place
It is incubated in incubator, sterilizing, obtain hair-keratin gel.
The invention provides a kind of dressing of wound repairing, its mainly have the advantage that for：
It is interrelated, synergistic between keratin and shitosan and other compositions in the present invention, make than independent
With having more excellent technique effect：It is in particular in the following aspects：(1) its collective effect makes that dressing is no any to exempt from
Epidemic focus, and do not find any toxic and side effect, even if allergic constitution also will not occur allergic phenomena；(2) have good
Biocompatibility, slowly can heal with wound and autologous skin growth, and hair-keratin can voluntarily dissolve and be absorbed by organisms, keep away
Exempt from the damage again that ordinary adjuvants stick together the surface of a wound, eliminate the misery of bleed during releasing many and patient, also will not leave behind chip and
Delay the healing of wound, the regeneration for promoting skin provides an environment being conducive to wound healing；(3) haemostatic effect is very
Good, and can effectively accelerate wound healing, and the partial action of skin barrier function can be played, it effectively stops bacterium infection；(4)
There is hygroscopicity, control and absorb wound exudate, keep the surface of a wound to be dried, promote cell growth, the effect of wound healing, keep away
Exempt from the damage again that ordinary adjuvants stick together the surface of a wound, eliminate the misery of bleed during releasing many and patient, also will not leave behind chip and
Delay the healing of wound, the regeneration for promoting skin provides an environment being conducive to wound healing；(5) gas permeability is strong, is suitable for
Gas and vapor pass through, the advantage such as (6) protection cambium.
Described by the method for the present invention (including raw material preparation, charging sequence, immersion and drying means number of times etc.)
The keratin of preparation, chitosan-based composite dressing for medical use are in porous support, and porous support assumes lamella loose structure, has higher
Porosity, suitable space structure can be provided to cell growth and migration.And this porous support is to Escherichia coli and golden yellow
Color staphylococcus has obvious inhibitory action, and porous support can promote erythrocyte aggregation to form aggregation, blocks blood vessel and breaks
The purpose of hemostasis is reached at damage；Meanwhile, porous support has good cell compatibility.Therefore, collagen/chitosan base is double-deck
Wound dressing has broad application prospects in terms of Wound healing and bone regeneration.
Brief description
Fig. 1 is the structural representation in light Microscopic observation hair-keratin described in the embodiment of the present invention；
Fig. 2 is in ESEM (1.69 × 10 described in the embodiment of the present invention3) under observe hair-keratin structure show
It is intended to；
Fig. 3 is in ESEM (1.25 × 10 described in the embodiment of the present invention3) under observe hair-keratin structure show
It is intended to；
Fig. 4 is in transmission electron microscope (1 × 10 described in the embodiment of the present invention5) under observe hair-keratin structural representation
Figure；
Fig. 5 is in transmission electron microscope (2 × 10 described in the embodiment of the present invention5) under observe hair-keratin structural representation
Figure；
Fig. 6 is the proterties of the hair-keratin of the gelation of 4% described in the embodiment of the present invention；
Fig. 7 is cutting into slices and immunohistochemistry dye for the wound tissue local repaired after damaging described in the embodiment of the present invention
Color；
Fig. 8 is, described in the embodiment of the present invention, keratin, chitosan-based composite dressing for medical use are fitted in showing of the rat surface of a wound
It is intended to；
Fig. 9 is the schematic diagram of Rat Wound Healing situation behind described in the embodiment of the present invention 21 days.
Specific embodiment
Purpose, technical scheme and advantage for making the embodiment of the present invention are clearer, specifically real below in conjunction with the present invention
Apply example technical scheme to be clearly and completely described it is clear that described embodiment is that a present invention part is real
Apply example, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creation
Property work under the premise of the every other embodiment that obtained, broadly fall into the scope of protection of the invention.
Embodiment 1
A kind of keratin, chitosan-based composite dressing for medical use, the composition of this dressing and the quality of various composition press following matter
Amount percentage adds：Keratin：2%；Sodium carboxymethylcellulose：1%；Shitosan：0.3%；Glycerine：8%；Balance of water.Its
In, keratin is the granular keratin prepared by human hair, and granular keratin is the keratin of powder.
Embodiment 2
A kind of keratin, chitosan-based composite dressing for medical use, the composition of this dressing and the quality of various composition press following matter
Amount percentage adds：Keratin：8%；Sodium carboxymethylcellulose：0.6%；Shitosan：0.9%；Glycerine：6%；Balance of water.
Wherein, keratin is the granular keratin prepared by wool, and granular keratin is the keratin of powder.
Embodiment 3
A kind of keratin, chitosan-based composite dressing for medical use, the composition of this dressing and the quality of various composition press following matter
Amount percentage adds：Keratin：4%；Sodium carboxymethylcellulose：0.9%；Shitosan：0.6%；Glycerine：6.5%；Balance of
Water.
Wherein, keratin is the keratin of the gelation being 3% by concentration prepared by wool, this gelation keratic
Preparation method is：Take in the water adding 5ml in the granular Keratin 20 0mg being prepared by wool, fully mix, be positioned over temperature
It is incubated at 37 DEG C in case, to assuming more homogeneous color and proterties；Sterilizing, obtains the hair-keratin of gelation,
Its concentration is the proterties of 4% hair-keratin of gelation for concentration for 4%, Fig. 6.
Embodiment 4
A kind of keratin, chitosan-based composite dressing for medical use, the composition of this dressing and the quality of various composition press following matter
Amount percentage adds：Keratin：6%；Sodium carboxymethylcellulose：0.8%；Shitosan：0.7%；Glycerine：7.5%；Balance of
Water.
Wherein, keratin is the keratin of the gelation being 2.5% by concentration prepared by human hair, the angle egg of this gelation
White preparation method is：Take and added in the water of 5ml by granular Keratin 1 25mg prepared by human hair, fully mix, place
It is incubated at 37 DEG C in incubator, to assuming more homogeneous color and proterties；Sterilizing, obtains the hair angle egg of gelation
In vain, its concentration is 2.5%, and the gelation hair-keratin of this concentration is the hair angle of 4% gelation than the concentration shown in Fig. 6
Albumen is diluter.
Embodiment 5
A kind of keratin, chitosan-based composite dressing for medical use, the composition of this dressing and the quality of various composition press following matter
Amount percentage adds：Keratin：5%；Sodium carboxymethylcellulose：0.8%；Shitosan：0.6%；Glycerine：7%；Water 86.6%.
Wherein, keratin is the keratin of the gelation being 2% by concentration prepared by human hair, the keratin of this gelation
Preparation method be：Take and added in the water of 5ml by granular Keratin 10 0mg prepared by human hair, fully mix, be positioned over
It is incubated at 37 DEG C in incubator, to assuming more homogeneous color and proterties；Sterilizing, obtains the hair angle egg of gelation
In vain, its concentration is 2%, and the gelation hair-keratin of this concentration is the hair angle egg of 4% gelation than the concentration shown in Fig. 6
Diluter in vain.
For the keratin described in any one embodiment in embodiment 1～embodiment 5, chitosan-based composite dressing for medical use, its
Composition also includes CaCl2And NaOH.
Embodiment 6
A kind of described keratin, the preparation method of chitosan-based composite dressing for medical use, comprise the following steps that：
S1：Prepare keratin；
S2：Then the sodium carboxymethylcellulose of the keratin of 5kg and 0.8kg is added in the water of 86.4kg, be designated as just molten
Liquid；
S3：The glycerine of the shitosan of 0.8kg and 7kg is added to the first solution of step S2, after mixing, pours training into
In foster ware, carry out first time freeze-drying at -20 DEG C, freeze-drying 2h about, that is, freeze-drying to water content is 20% left side
Till the right side；
S4：Material after first time freeze-drying in step S3 is put into the CaCl that concentration is 0.5%2Soak in solution
Bubble 2h, from CaCl2Immersion 3h in the NaOH solution that concentration is 1% is placed into, immersion finishes after taking out material in solution ,-
Carry out second freeze-drying at 30 DEG C, freeze-drying 5h about, that is, freeze-drying is 10% about to water content；
S5：Again material is put into leaching in the glycerine water solution that concentration is 20% after second freeze-drying of step S4
Bubble 2h, immersion finishes, and carries out third time freeze-drying at -50 DEG C, freeze-drying 12h about, that is, freeze-drying is to water content
Till 3%, that is, obtain keratin, chitosan-based composite dressing for medical use.
In step S1, keratin is granular keratin, and granular keratic concrete preparation process is as follows：
①：Degreasing：Weigh 5g human hair, be placed in 1000ml beaker.Add 99.7% soaked in absolute ethyl alcohol 30 minutes,
Stirred once with glass bar every 5 minutes, flowing water rinses；
②：Soften：Hair after cleaning is positioned over >=10% (mass volume ratio) sodium hypochlorite 10ml and distilled water
In the solution of 90ml configuration, soak at room temperature 30 minutes, interval is stirred once for 5 minutes with glass bar, and flowing water rinses；
③：Decolouring：By the weight of 5g hair, weigh 99% sodium pyrophosphate (Na4P2O7) (mass volume ratio) 4g, 99.5%
Potassium peroxydisulfate (K2S2O8) (mass volume ratio) 1.5g, 30% hydrogen peroxide (H2O2) 80ml (percent by volume v/v), 25% concentrated ammonia liquor
27.5ml (percent by volume v/v), distilled water 400ml, are configured to de-inking solution, the hair of softening is placed in this solution, puts
It is placed in 37 DEG C of Water Tank with Temp.-controlled, was stirred 1 time with glass bar every 20 minutes, observe hair color to light yellow, this process
Used time general 2-3 hour is short more a lot of than conventional method；Hair after decolouring is placed on screen cloth, flowing water rinses, until washing
Fall de-inking solution.
④：Cleaning, drying：Remove the hair more than 12 hours after ionized water soaks above-mentioned decolouring, remove remaining reagent；Go
Ionized water rinses, and is placed in air dry oven 37 DEG C and dries overnight after filtration；
⑤：Grind：It is placed in ceramic mortar after Hair grooming scissors after drying is shredded, add liquid nitrogen frozen to grind, extremely
Hair becomes 1mm length, obtains hair meal；
⑥：Ball milling：Weigh hair meal 2g, be placed in ball grinder, put into agate bead, add physiological saline 10ml, 460
Turn/min, ball milling 2 hours, be inverted the size of observed under electron microscope particle diameter, add physiological saline 5ml and continue grinding 1.5
Individual hour, until form human hair's keratin particle homogenate of about 70 μm of particle diameter；The quantity of above-mentioned agate bead：Diameter 1cm agate
20, Nao pearl, diameter 6mm agate bead 100；
⑦：Tubulature：In above-mentioned homogenate add physiological saline 30ml, by the liquid rinse in ball grinder be transferred to 50ml from
In heart pipe；
⑧：Purify：Centrifuge tube is placed in the Centrifuge Cup of centrifuge, 3000 turns/min of centrifugal speed, is centrifuged 20min, abandons
Remove supernatant, remove the top layer of keratin precipitation and bottom with key, take the keratin of stage casing even particle size move to new from
In heart pipe, plus physiological saline 40ml mixes, and continues centrifugation once, abandoning supernatant, then removes the table of keratin precipitation with key
(key can also be substituted for some similar glass bars or sheet glass etc. to layer, as long as upper and lower two parts are scraped off just with bottom
Can, it is easily accomplished in practical operation), take the keratin of stage casing even particle size to move in new centrifuge tube, then plus physiological saline
40ml mixes, and is centrifuged again, abandons supernatant, then the top layer with key removal keratin precipitation and bottom, takes stage casing granular size
Uniform keratin moves in new centrifuge tube, obtains highly purified granular hair-keratin, because particle diameter is little, therefore takes a fancy to
Go as keratin mud；It is temporarily placed in 4 DEG C to save backup；
⑨：Sterilizing：The highly purified granular hair-keratin obtaining is loaded in polybag, good seal, uses carton
Deliver to radiation center after packaging and carry out Co60Radiation sterilization, dosage 12Kg, it is placed on 4 DEG C of refrigerators, stand-by；
The highly purified granular hair-keratin preparing is observed under om observation (× 20), it maintains hair
The fascicular texture that to send out original stable, as shown in Figure 1.
Fig. 2～5 are this granular hair-keratin electron microscope, and Fig. 2 behaves and acts in an unreasonable way section SEM (1.69 × 103), Fig. 3
Face SEM (1.25 × 10 is delivered in behaviour3), Fig. 4 is human hair TEM (1 × 105), Fig. 5 is human hair cortex TEM (2 × 105).
From ESEM (SEM), the Hair-material surface smoother processing through the inventive method, no normal dark hair
Scale structure, the convex patch shape in scrobicula；Cross section hair cortex is still in no structure homogeneous shape.By the visible hair of transmission electron microscope (TEM)
Send out melanin in keratin granular materials to be all disintegrated.
It should be noted that the human hair that the present embodiment is used is changed to wool, related parameter, step etc. are all with adopting
Prepare keratin with human hair consistent.
Embodiment 7
A kind of described keratin, the preparation method of chitosan-based composite dressing for medical use, comprise the following steps that：
S1：Prepare keratin；
S2：Then the sodium carboxymethylcellulose of the keratin of 3kg and 0.7kg is added in the water of 88.9kg, be designated as just molten
Liquid；
S3：The glycerine of the shitosan of 0.9kg and 6.5kg is added to the first solution of step S2, after mixing, pours into
In culture dish, carry out first time freeze-drying at -23 DEG C, freeze-drying 3h about, that is, freeze-drying to water content is 20%
Till；
S4：Material after first time freeze-drying in step S3 is put into the CaCl that concentration is 0.85%2In solution
Soak 3h, from CaCl2Immersion 2h in the NaOH solution that concentration is 1.75% is placed into, immersion finishes after taking out material in solution
Carry out second freeze-drying again at -35 DEG C, freeze-drying 4h about, that is, freeze-drying is 12% to water content；
S5：Again material is put into leaching in the glycerine water solution that concentration is 30% after second freeze-drying of step S4
Bubble 4h, immersion finishes and carries out third time freeze-drying again at -50 DEG C, freeze-drying 10h, and freeze-drying to water content is 5%
Till, that is, obtain keratin, chitosan-based composite dressing for medical use.
In step S1, keratin is the keratin of gelation, and the keratic concrete preparation process of gelation is as follows：
(1) prepare granular keratin, preparation method with step in embodiment 6 1.～9. in granular keratic
Preparation method, is just not repeated explanation here；
(2) take in the water adding 5ml in obtained highly purified granular Keratin 20 0mg in step (1), fully
Mix, be positioned in incubator and be incubated at 37 DEG C, to assuming more homogeneous color and proterties；Sterilizing, obtains gelation
Hair-keratin, its concentration is the proterties of 4% hair-keratin of gelation for concentration for 4%, Fig. 6.
It should be noted that the human hair that the present embodiment is used is changed to wool, related parameter, step etc. are all with adopting
Prepare keratin with human hair consistent.
Embodiment 8
A kind of described keratin, the preparation method of chitosan-based composite dressing for medical use, comprise the following steps that：
S1：Prepare keratin；
S2：Then the sodium carboxymethylcellulose of the keratin of 7kg and 0.9kg is added in the water of 84.1kg, be designated as just molten
Liquid；
S3：The glycerine of the shitosan of 0.5kg and 7.5kg is added to the first solution of step S2, after mixing, pours into
In culture dish, carry out first time freeze-drying at -15 DEG C, freeze-drying 3h about, that is, freeze-drying to water content is 18%
Till；
S4：Material after first time freeze-drying in step S3 is put into the CaCl that concentration is 0.75%2In solution
Soak 3h, from CaCl2Immersion 2h in the NaOH solution that concentration is 1.5% is placed into, immersion finishes after taking out material in solution
Carry out second freeze-drying again at -35 DEG C, freeze-drying 4h about, that is, freeze-drying is 12% to water content；
S5：Again material is put into leaching in the glycerine water solution that concentration is 25% after second freeze-drying of step S4
Bubble 4h, immersion finishes and carries out third time freeze-drying again at -55 DEG C, freeze-drying 15h about, that is, freeze-drying is to water content
Till 2%, that is, obtain keratin, chitosan-based composite dressing for medical use.
In step S1, keratin is the keratin of gelation, and the keratic concrete preparation process of gelation is as follows：
S1：Weigh 10g wool, put into the there-necked flask of the mixed solution equipped with 180ml absolute ethyl alcohol and 20ml distilled water
In, heating was warming up to 75 DEG C after 15 minutes, and solution starts to flow back；Then, drip the NaOH solution of 2ml in backflow, to oxidation
Keratic pH value is 7, and backflow is terminated with dropping simultaneously, and the time continues 90 minutes；Dropping and after backflow terminates, continues at 25 DEG C
Stirring 5 hours, makes reaction completely, obtains into salt hair-keratin.
S2：Salt hair-keratin will be become to leach, and rinsed with washing lotion, this washing lotion is that absolute ethyl alcohol is mixed with equivalent distilled water
Close, remove unreacted NaOH, with pH detection paper whether in alkalescence, wash to not being in alkalescence, put into 60 DEG C of air dry ovens
Middle drying 6 hours, obtains the hair-keratin sulfonate of drying；
S3：It is in fragility through hair-keratin sulfonate obtained from above-mentioned process, in Meteor-style ball grinder, put into agate
Nao pearl, grinds under conditions of 460 turns/min, till pulverizing as 50 μm about of hair-keratin sulfonic acid salt powder, is lyophilized；
The quantity of agate bead can be：Diameter 1cm agate bead 80, diameter 6mm agate bead 300；
S4：The hair-keratin of gelation：Operate in super-clean bench, take obtain in 100mg step S3 lyophilized after hair
Send out and in keratin sulfonic acid salt fines, add 5ml sterilized water, first adopt agitator to mix material by hand, more fully mixed with mediation mixing machine
Even, it is positioned over 37 DEG C of incubations about 3 hours, then obtain color and the more homogeneous hair-keratin gel of proterties, then, adopt
With the sterilizing of 60Coradiation method, obtain the hair-keratin of gelation, its concentration is 2%, and the hair-keratin of this gelation compares Fig. 6
The hair-keratin of shown gelation is diluter.
It should be noted that the wool that the present embodiment is used is changed to human hair, related parameter, step etc. are all with adopting
Prepare keratin with human hair consistent.
Embodiment 9
Biocompatibility and toxicity：
Experimental group：1. the keratin prepared by the present invention of 100% concentration, chitosan-based composite dressing for medical use PBS/ physiology salt
Water extract；2. the keratin of 100% concentration, chitosan-based composite dressing for medical use PBS/ physiological saline leaching liquor and DMEM are cultivated
Base 1:1 volume mixture is diluted to 50% concentration；3. by the keratin of 100% concentration, chitosan-based composite dressing for medical use PBS/ physiology
Salt water extract and DMEM culture medium 1:3 volume mixture are diluted to 25% concentration；
Control group：PBS/ physiological saline.
Method：Collect logarithmic phase L-929 cell, with 5 × 104Individual/ml density is inoculated in 96 orifice plates, every hole 100 μ l, is divided into
4 groups (3 concentration group+control groups), every group of repetition 8 holes.Put 37 DEG C, 5%CO2Incubator is cultivated 24 hours, discards stoste, every hole
Change to the new nutrient solution of 100 μ l, every group of each hole of experimental group is separately added into accordingly being subject to of above-mentioned 100%, 50%, 25% of 100 μ l again
Test solution, control group adds the physiological saline of same amount.Put 37 DEG C, 5%CO2Incubator was cultivated, in the 3rd day of culture, the 5th day and the 7th
It takes out one piece of 96 orifice plate respectively, discards every hole supernatant 110 μ l, adds 10 μ l MTT (0.5%MTT, 3- (4,5- dimethyl thiophenes
Azoles -2) -2,5- diphenyltetrazolium bromide bromide, i.e. tetrazolium bromide), put in case and cultivate 4 hours, take plate, carefully suck in the hole supernatant, often
Hole adds DMSO (dimethyl sulfoxide (DMSO)) 150 μ l, places into 37 DEG C, 5%CO2It is incubated in incubator 15 minutes, measure luminosity as early as possible
Value (OD value), mensure wavelength is 490nm, and reference wavelength is 540nm.
Calculate cell by formula (RGR=material each concentration group absorbance/negative control group absorbance × 100%) relatively to increase
Degree of growing (RGR), and it is converted into cytotoxicity classification according to cell relative to the relation of the rate of increase and cytotoxicity classification.Result table
Bright, the cell that the medical dressing of the application of variable concentrations records in three time points all reaches more than 80% relative to the rate of increase, carefully
Cellular toxicity is classified as one-level, illustrates there is good cell compatibility, no cytotoxicity.As shown in table 1.
Table 1：MTT result statistical analysis
Embodiment 10
Take healthy SD rat, anaesthetized with pentobarbital (30mg/kg), open 1cm by its back backbone side, scraper holostrome is cut
Except skin, form diameter 1.8cm, an area 2.54cm2The circular full thickness skin excision surface of a wound, offside antimere skin makees
For normal own control.Dressing patch prepared by the present invention, together in the surface of a wound, makes the keratin layer of dressing and the surface of a wound fit tightly, such as
Shown in Fig. 8.
Observe：The rat surface of a wound covers 10 minutes about after dressing, and bleeding tapers off, the 3d surface of a wound after test group of animals wound
Reduce it is seen that new granulation tissue generates.After wound during 21d, more than 90% wound healing, as shown in Figure 9.
It should be noted that HE staining tissue slides are observed：Survey skin corium to thicken, average thickness about 0.58mm when 4 weeks, can
See and assemble under substantial amounts of keratin particle corium, have cell and fibrous tissue growth between keratin, have a small amount of multinucleate giant cell,
Interior phagocytosis has keratin particle.
Immunohistochemical staining is observed：In section, mouse dermal fibroblast secretes NTx albumen in a large number, and collagen is fine
Dimension arrangement is tight, brown stain, and NTx albumen and histocyte are interspersed；Filling position cutaneous immunisation group after 8th week
Weave chemistry dyes observation figure as shown in fig. 7, Fig. 7 is the dyeing display NTx PE situation of mouse dermal fibroblast,
It follows that display skin histology healing is good, that is, mouse skin wound recovery is good.
Bacterium infection is observed：Have no obvious bacterium infection situation injured to after fully recover.
Conclusion：The based composite dressing for medical use effect highly significant of the present invention, in the case of not having any medication, does not occur bright
21d after aobvious inflammation, and wound, most wound healings, hour angle protein gel can be degraded substantially within 12 weeks.
Specifically, the based composite dressing for medical use in the present invention has the porous support knot penetrating into beneficial to cell, adhering to and breed
Structure, has the characteristic being substantially better than other materials in terms of cell adherence and propagation, has nerve-inducing, in hemostasis side simultaneously
Face also shows that good effect；Also illustrate that the present invention passes through the effect pole of hair-keratin and the synergistic application such as shitosan
It is notable.
Note：The studies above is all carried out in the case of Ethics Committee and experimenter's informed consent.
When being embodied as, in the present invention, for any embodiment in embodiment 1～5, if CaCl to be added2And NaOH,
Then for CaCl2Do not specially require with the amount of NaOH, CaCl2As long as add with the amount of NaOH no longer reacting to other compositions
Till.
Percentage composition used in the present invention is the conventional percentage composition in market.
Finally it should be noted that：Above-described each embodiment is merely to illustrate technical scheme, rather than to it
Limit；Although being described in detail to the present invention with reference to the foregoing embodiments, it will be understood by those within the art that：
It still can be modified to the technical scheme described in previous embodiment, or wherein part or all of technical characteristic is entered
Row equivalent；And these modifications or replacement, do not make the essence of appropriate technical solution depart from various embodiments of the present invention technical side
The scope of case.
Claims (10)
1. a kind of keratin, chitosan-based composite dressing for medical use it is characterised in that：The material composition of described dressing and various composition
Mass percent be：Keratin：2%～8%；Sodium carboxymethylcellulose：0.6%～1%；Shitosan：0.3%～0.9%；
Glycerine：6%～8%；Balance of water.
2. keratin according to claim 1, chitosan-based composite dressing for medical use it is characterised in that：The raw material of described dressing
The mass percent of composition and various composition is：Keratin：4%～6%；Sodium carboxymethylcellulose：0.8%～0.9%；Shell
Glycan：0.6%～0.7%；Glycerine：7%；Balance of water.
3. keratin according to claim 2, chitosan-based composite dressing for medical use it is characterised in that：Described keratin is hair
Send out keratin, the material composition of described dressing also includes CaCl2Solution, NaOH solution and glycerine water solution.
4. a kind of keratin according to any one of claims 1 to 3, the preparation method of chitosan-based composite dressing for medical use,
It is characterized in that：Described preparation method is as follows：
S1：Prepare keratin；
S2：Weigh various composition in proportion, then keratin and sodium carboxymethylcellulose are added to the water to obtain first solution；
S3：In first solution described in shitosan and glycerine are added to step S2, after mixing, carry out first time freeze-drying；
S4：It is sequentially placed into CaCl after the first time freeze-drying of step S32Soak in solution and NaOH solution, immersion finishes
Carry out second freeze-drying again；
S5：Soak in glycerine water solution again after second freeze-drying of step S4, immersion finishes and carries out third time again
Freeze-drying, that is, obtain keratin, chitosan-based composite dressing for medical use.
5. keratin according to claim 4, chitosan-based composite dressing for medical use preparation method it is characterised in that：Step
Keratin described in S1 is hair-keratin, and described hair-keratin is the hair of granular hair-keratin and gelation
Any one in keratin, the concentration of the hair-keratin of described gelation is 0.5～5%.
6. keratin according to claim 5, chitosan-based composite dressing for medical use preparation method it is characterised in that：Described
The preparation method of granular hair-keratin is：1. degreasing and softening：Weigh hair and carry out degreasing and softening；2. decolour；③
Cleaning, drying：Hair after decolouring is carried out, and is dried；4. the preparation of particle：Dried hair is shredded, and
Add liquid nitrogen frozen to grind, obtain hair meal；Meal is placed in ball grinder, puts into agate bead, add physiological saline, rotation
Turn, ball milling, until particle diameter reaches 50 μm～90 μm, obtain the homogenate of hair-keratin particle；5. purify：Physiology is added in homogenate
Salt solution is centrifuged, abandoning supernatant, and removes top layer and the bottom of precipitation, takes the hair angle egg of stage casing even particle size
In vain, plus physiological saline mixes and continues centrifugation, repeat aforesaid operations 2～4 times, finally still take the hair of stage casing even particle size
Keratin, then obtain highly purified granular hair-keratin, standby after sterilizing.
7. keratin according to claim 6, chitosan-based composite dressing for medical use preparation method it is characterised in that：Step
2. described in, the detailed process of decolouring is：Hair after softening is positioned in de-inking solution and is decoloured, described de-inking solution
Composition be sodium pyrophosphate 3～5 weight portion, potassium peroxydisulfate 1～2 weight portion, hydrogen peroxide 70～90 parts by volume, concentrated ammonia liquor 22～33
Parts by volume and water 350～450 parts by volume.
8. keratin according to claim 6, chitosan-based composite dressing for medical use preparation method it is characterised in that：Step
4. detailed process is：Dried hair is shredded, and adds liquid nitrogen frozen to grind, obtain the hair that length is 0.1～2mm
Meal；Hair meal is placed in ball grinder, puts into agate bead, add the quality of physiological saline, hair meal and physiological saline
Volume ratio is 2:5, ball milling 1～3 hour, until form about 60 μm -80 μm of particle diameter；The quantity of described agate bead is：The hair of every 1g
Send out the agate bead 50 of the agate bead 10 and diameter 6mm of the corresponding diameter 1cm of meal.
9. keratin according to claim 6, chitosan-based composite dressing for medical use preparation method it is characterised in that：Described
The preparation method of the hair-keratin of gelation is：Take step 5. in prepared granular hair-keratin, add water, fully
Mix, be positioned over incubation in incubator, to assuming more homogeneous color and proterties, obtain gelatinous hair-keratin,
Standby after sterilizing.
10. the wound repairing according to any one of claim 5～9 dressing it is characterised in that：The hair of described gelation
Send out keratin can also be prepared by the following method：
(1) hair-keratin becomes salt：Weigh hair, add the mixed solution of second alcohol and water, heating, backflow, drip in backflow
Aqueous slkali, is to terminate when 6.5～7.5 to pH value, then proceedes to stir, make reaction completely, be filtrated to get hair-keratin salt, do
Dry, it is crushed to micron order, be lyophilized, that is, obtain hair-keratin sulfonic acid salt powder；
(2) preparation of hair-keratin gel：Weigh hair-keratin sulfonic acid salt powder, add water, fully mix, be positioned over temperature
It is incubated in case, sterilizing, obtain hair-keratin gel.
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| CN112156224A (en) * | 2020-01-20 | 2021-01-01 | 海南海默斯医学生物科技有限公司 | Composition for preparing keratin liquid dressing and preparation method and application thereof |
| CN112245653A (en) * | 2020-11-13 | 2021-01-22 | 杭州伊瑟奇生物科技有限公司 | Protein film dressing assisting wound healing based on cooperation of three bioactive scaffold materials and cell trophic factors |
| CN112516305A (en) * | 2019-08-28 | 2021-03-19 | 天津大学 | Preparation method of human hair melanosome derivative and application of human hair melanosome derivative in antibiosis and tissue repair |
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| US8273702B2 (en) * | 2006-02-17 | 2012-09-25 | Wake Forest University Health Sciences | Wound healing compositions containing keratin biomaterials |
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| CN112245653A (en) * | 2020-11-13 | 2021-01-22 | 杭州伊瑟奇生物科技有限公司 | Protein film dressing assisting wound healing based on cooperation of three bioactive scaffold materials and cell trophic factors |
| CN112245653B (en) * | 2020-11-13 | 2022-07-01 | 杭州伊瑟奇生物科技有限公司 | Protein film dressing assisting wound healing based on cooperation of three bioactive scaffold materials and cell trophic factors |
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