CN104189945A - Double-layered composite biological dressing and adhesive-free compound technology thereof - Google Patents

Double-layered composite biological dressing and adhesive-free compound technology thereof Download PDF

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CN104189945A
CN104189945A CN201410508777.9A CN201410508777A CN104189945A CN 104189945 A CN104189945 A CN 104189945A CN 201410508777 A CN201410508777 A CN 201410508777A CN 104189945 A CN104189945 A CN 104189945A
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chitosan
spongy
material solution
dressing
layer material
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CN104189945B (en
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郭养浩
郑允权
石贤爱
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Fuzhou University
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Fuzhou University
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Abstract

The invention provides a double-layered composite biological dressing and an adhesive-free compound technology thereof. The double-layered composite dressing comprises a chitosan fiber non-woven fabric layer and a spongy-shaped functional layer, wherein no adhesive is needed to bond the two layers. The preparation technology comprises the following steps: (1) chitosan fiber non-woven fabric is pre-cooled; (2) macromolecule chitosan, macromolecule collagen protein, micromolecule collagen protein and micromolecule chitosan in mass ratio of 0.7-1.3 to 0.1-0.3 to 0.7-1.3 to 0.1-0.3 are added in a faintly acid aqueous solution to prepare a spongy layer material solution; (3) the pre-cooled chitosan non-woven fabric is taken out and the spongy layer material solution is rapidly and uniformly sprayed on the non-woven fabric, wherein the spraying amount is 20-60 ml/20*20 cm<2>; (4) the chitosan non-woven fabric sprayed with the spongy layer material solution is freeze-dried. The prepared double-layered composite dressing is firm in embedding connection between the two layers; the spongy functional layer has the function of promoting wound healing and prevents adhesion; the chitosan fiber non-woven fabric layer has the functions of favorable air permeability, comfort and bacterium resistance.

Description

A kind of two-layer compound biological dressing and without adhesive combination process
Technical field
The invention belongs to the preparation field of medical wound dressing, be specifically related to a kind of two-layer compound biological dressing and without adhesive combination process.
Background technology
Skin suffers after wound or burn. conventionally need to, with a kind of wound dressing protection wound surface of function admirable, provide an environment that is conducive to wound healing.And for above wound or the burn of dark two degree, except needing dressing that necessary protecting wound and antibacterial functions is provided, also need dressing to possess and can promote remaining epithelium regeneration and accelerate the function of wound healing, until rebuild permanent skin barrier.
Chitosan has good protecting and creates anthemorrhagic performance and bacteriostasis property, and biocompatibility is strong, and chitin fiber non-woven fabrics can be used as wound dressing.But because chitin fiber is easy and wound sticks together, the more newly-generated granulation of easy damaged wound when change dressings, and cause patient suffering, therefore conventional chitin fiber wound dressing surface needs to adopt chemical viscose agent to stick adherence preventing material, as polyester antiblocking layers.The existence of antiblocking layers has reduced the probability that wound and fiber stick together, but in wound microenvironment, multiple beneficial effect of chitosan cannot play a role.Moreover for deep burn or degree of depth surgical wound wound, synthetic macromolecule antiblocking layers is inapplicable.
Collagen sponge is the biological dressing with spongy loose structure of being made up of collagen protein, conventionally adopts freeze-drying preparation.Simple collagen sponge is easily dry and cracked, and degradation rate is too fast, mechanical strength is little, by natural polysaccharide compound such as collagen protein and chitosan, hyaluronic acids, can improve to a certain extent its performance.Collagen sponge has good hydrophilic and water absorption, and its adhesiveness is good, can the long period cover for wound surface; Be applicable to deep wounds, there is effect of accelerating wound healing, but it lacks the effect of protecting bacteria infection in wound and isolation (or inhibition) air, when clinical use, need add a cover multiple ply sterilization gauze.
It is bonding that the preparation technology of published compound dressing all adopts chemical adhesive to carry out.Patent of invention 201310539212.2 discloses a kind of preparation method of the compound chitosan dressing being made up of the short more layer of chitosan and chitosan hemostatic layer, hemostatic layer is chitosan particle, and the formation of hemostatic layer and hemostatic layer and the short more formation of layer are all passed through glue bond.Utility model patent 201220613590.1 adopts the bonding method of binding agent to prepare a kind of two-layer compound bio dressing, and described upper strata polyurethane film is the thin film that scribbles binding agent, is affixed on the PVA/PVP/ of lower floor carboxymethyl chitosan/shell iodine material.Adopt adhesive adhesion complex method, adhesive is introduced in dressing, brought unnecessary composition to increase and potential harm.
To there is the collagen sponge that promotes wound healing function and there is the chitosan dressing that protects wound bacteria resistance function and carry out compoundly, the performance of combine dressing can be brought up to comparatively desirable state.A wound dressing for function admirable, learns performance and physical and chemical performance if having good biological, easy to use, and does not contain unnecessary chemical composition, as adhesive etc., has application prospect widely.
Summary of the invention
The object of the invention is to the some shortcomings that exist for existing dressing, provide a kind of two-layer compound biological dressing and without adhesive combination process.Prepared two-layer compound dressing is chimeric between layers, and firmly spongy functional layer has the function that promotes wound healing, can anti; Chitin fiber nonwoven layer breathability and comfortableness are good, and have good antibacterial functions.
For realizing the object of the invention, adopt following technical scheme:
A kind of two-layer compound biological dressing, by chitin fiber non-woven fabrics and spongy layer material solution, under the condition that does not add adhesive, low-temperature freeze drying is chimeric forms in described dressing, and lower floor is chitin fiber nonwoven layer, and upper strata is spongy functional layer; In described spongy layer material solution, contain macromole chitosan, micromolecule chitosan, macromolecular collagen protein, micromolecular collagen.
Chimeric thickness between the upper and lower of combine dressing is 30-120 μ m.
A technique of preparing described two-layer compound biological dressing, comprises the following steps:
(1) after chitin fiber nonwoven surface spray pure water, freeze at-20 ~-40 DEG C;
(2) preparation spongy layer material solution, after stirring, ultrasonic degas;
(3) the chitin fiber non-woven fabrics of taking-up prefreezing, is evenly sprayed at spongy layer material solution on non-woven fabrics rapidly;
(4) by the chitin fiber non-woven fabrics that is coated with spongy layer material solution-40 ~-50 DEG C freezing after, vacuum drying makes two-layer compound biological dressing.
In described spongy layer material solution in step (2), the quality proportioning of macromole chitosan (molecular weight is greater than 50,000 Da), micromolecule chitosan (mean molecule quantity 2000Da), macromolecular collagen protein (molecular weight is greater than 80,000 Da), micromolecular collagen (mean molecule quantity 1000Da) is: 0.7-1.3:0.1-0.3:0.7-1.3:0.1-0.3, and total concentration is 2.0-3.5wt%;
In described step (3), the spray flux of spongy layer material solution is 20-60ml/20*20cm 2;
Chitin fiber nonwoven is furnished with dividing of acupuncture and water thorn, plain edition (hydrophobic) and modified model (easily water suction), first-selected plain edition (hydrophobic) acupuncture of the present invention chitin fiber non-woven fabrics.
Chitin fiber nonwoven surface has space between the fiber varying in size (0.01-1.0mm), and the speed that aqueous solution penetrates into non-woven fabrics gap depends on the size in space between the hydrophilic of non-woven fabrics and fiber.Excessive spongy layer material solution infiltrates through lower floor's chitosan non-woven fabrics, not only causes the functional layer inventory of being close to wound to decline, and causes the hardening of final freeze-drying prods quality, and comfortableness and permeability significantly decline, and anti-microbial property is corresponding decline also.The present invention, by chitosan nonwoven surface spray suitable quantity of water and carry out the method for pre-freeze, can seal the large space between non-woven fabrics fiber, for the infiltration of controlling appropriate spongy layer material solution provides condition.
The invention provides a kind of course control method for use of controlling combine dressing hybrid layer thickness.Can control combine dressing hybrid layer thickness to the strict control of relevant parameter.Suitable hybrid layer thickness both can ensure that double-deck dressing, in conjunction with firmly, can ensure that again combine dressing levels is all in the required optimum state of medical dressing.
Useful technique effect of the present invention is:
1 the invention provides one without adhesive two-layer compound biological dressing, has the active layers of two different composition 26S Proteasome Structure and Functions, double-deck knot
Close firmly; Lower floor is that chitin fiber protects wound antibiotic layer, keeps the original breathability of chitosan non-woven fabrics, comfortableness and good anti-microbial property; Upper strata is the spongy functional layer of collagen protein and chitosan, and flexible, absorption speed is fast, has the function of wound healing of promotion, can anti;
The present invention provide a kind of without the bonding double-deck dressing combination process of adhesive.Spongy functional layer chimeric enter chitin fiber nonwoven layer, the about 30-120 μ of hybrid layer thickness m, can ensure that combine dressing levels is all in the required optimum state of medical dressing;
3. the invention provides a kind of course control method for use of controlling combine dressing hybrid layer thickness, spongy layer material solution depends on the infiltration capacity of chitin fiber: the performance of chitin fiber non-woven fabrics and spongy layer material solution arrive the behavior after the non-woven fabrics of pre-freeze at spray, non-woven fabric freezing water quantity and prefreezing temperature, spray the spongy layer material solution temperature on non-woven fabrics, volume and concentration, pre-freeze non-woven fabrics taking-up operation and the again time difference of refrigeration operation, aforesaid operations parameter will directly affect time of penetration and the length of penetration of spongy layer solution to lower floor's chitin fiber.Can control combine dressing hybrid layer thickness to the strict control of above-mentioned parameter, thereby guarantee the superperformance of double-deck dressing;
4. sponge functional layer of the present invention contains macromole chitosan, micromolecule chitosan, macromolecular collagen protein, micromolecular collagen, and its quality proportioning is: (0.7-1.3)/and (0.1-0.3)/(0.7-1.3)/(0.1-0.3).Wherein macromolecular collagen protein and macromole chitosan form the skeleton of spongy layer jointly, and micromolecular collagen and micromolecule chitosan are dissolved in macromolecular skeleton structure; The function that the relative stability of spongy layer framing structure is conducive to keep high water absorbing properties under a large amount of wound sepage conditions and covers wound; Micromolecular collagen and micromolecule chitosan are extracted into wound surface, bring into play effect antibacterial and promotion wound healing;
5. two-layer compound biological dressing of the present invention can be used for above burn or the surgical wound of dark two degree, has following feature: (1) good biocompatibility, be closely affixed with wound, and there is the good wound function of protecting; (2) bacteria growing inhibiting, prevent wound infection; (3) promote wound skin flbroblast growth, accelerating wound; (4) use safety, no cytotoxicity, without chemical adhesive potential hazard; (5) water absorption, breathability, comfortableness are good; (6) application is convenient, is not adhered and causes secondary injury.
Brief description of the drawings
Fig. 1 is the electron-microscope scanning figure in combine dressing cross section.
Detailed description of the invention
The present invention further illustrates the present invention with the following example, but protection scope of the present invention is not limited to the following example.
Embodiment 1
By 20*20cm 2100g/m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 30ml RO pure water, be placed in ultra cold storage freezer ,-40 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 3.5wt%, take macromole chitosan 1.1g, micromolecule chitosan 0.3g, macromolecular collagen protein 1.1g, micromolecular collagen 0.3g, be dissolved in the aqueous acetic acid of 80ml 0.5wt%, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 40ml/20*20cm 2, the operating time is less than 5min; Put into ultra cold storage freezer ,-50 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 10h makes without adhesive two-layer compound biological dressing;
Prepared casting product is double-deck, and in conjunction with firm, hybrid layer thickness is 30-80 μ m, the about 450-550 μ of the spongy functional layer thickness in upper strata m, and quality is slightly partially hard, flexible, and absorption speed is fast; Dressing overall air permeability, water absorption and comfortableness are better, and anti-microbial property is strong;
Control experiment, above-mentioned spongy layer material solution is sprayed in not pretreated nonwoven surface, under follow-up same operation condition, prepared dressing has no obvious hybrid layer, upper strata sponge-like layer thickness less than 50 μ m, a large amount of spongy layer material solutions infiltrate lower floor's non-woven fabrics, cause lower floor's non-woven fabrics quality hardening, and dressing overall air permeability and comfortableness have been not suitable as medical dressing.
Embodiment 2
By 20*20cm 2100g/ m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 40ml RO pure water, be placed in ultra cold storage freezer ,-40 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 2.5wt%, take macromole chitosan 0.8, micromolecule chitosan 0.2g, macromolecular collagen protein 0.8g, micromolecular collagen 0.2g, be dissolved in the aqueous acetic acid of 80ml 0.5wt%, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 60ml/20*20cm 2, the operating time is less than 5min; Put into ultra cold storage freezer ,-40 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 12h makes without glue composite sponge dressing;
Prepared casting product is double-deck in conjunction with firm, hybrid layer thickness 60-120 μ m, and the about 700-800 μ of the spongy functional layer thickness in upper strata m, quality softness, flexible, absorption speed is fast; Dressing overall air permeability, water absorption and comfortableness are good, and anti-microbial property is strong;
Control experiment, above-mentioned spongy layer material solution is sprayed in not pretreated nonwoven surface, under follow-up same operation condition, prepared dressing has no obvious hybrid layer, upper strata sponge-like layer thickness less than 50 μ m, a large amount of functional layer material solutions infiltrate lower floor's non-woven fabrics, cause lower floor's non-woven fabrics quality hardening, and dressing overall air permeability and comfortableness have been not suitable as medical dressing.
Embodiment 3
By 20*20cm 2100g/ m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 30ml RO pure water, be placed in ultra cold storage freezer ,-30 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 2.0wt%, take macromole chitosan 0.8g, micromolecule chitosan 0.2g, macromolecular collagen protein 0.8g, micromolecular collagen 0.2g, be dissolved in 0.5% the aqueous acetic acid of 100ml, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 50ml/20*20cm 2, the operating time is less than 5min; Put into cryogenic refrigerator ,-40 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 11h makes without glue composite sponge dressing;
Prepared casting product is double-deck in conjunction with firm, hybrid layer thickness 60-110 μ m, and the about 550-600 μ of the spongy functional layer thickness in upper strata m, quality softness, flexible, absorption speed is fast.Dressing overall air permeability, water absorption and comfortableness are good, and anti-microbial property is strong.
Embodiment 4
By 20*20cm 2100g/ m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 30ml RO pure water, be placed in ultra cold storage freezer ,-20 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 3.0wt%, take macromole chitosan 0.7g, micromolecule chitosan 0.2g, macromolecular collagen protein 1.3g, micromolecular collagen 0.2g, be dissolved in the aqueous acetic acid of 80ml 0.5wt%, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 40ml/20*20cm 2, the operating time is less than 5min; Put into cryogenic refrigerator ,-40 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 10h makes without glue composite sponge dressing;
Prepared casting product is double-deck in conjunction with firm, the about 50-100 μ of hybrid layer thickness m, and the about 450-500 μ of the spongy functional layer thickness in upper strata m, quality is comparatively soft, and absorption speed is fast, but after inhaling a small amount of water, spongy layer skeleton is yielding.Dressing overall air permeability, water absorption and comfortableness are better, and anti-microbial property is strong.
Embodiment 5
By 20*20cm 2100g/ m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 30ml RO pure water, be placed in ultra cold storage freezer ,-30 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 3.0wt%, take macromole chitosan 1.3g, micromolecule chitosan 0.2g, macromolecular collagen protein 0.7g, micromolecular collagen 0.2g, be dissolved in the aqueous acetic acid of 80ml 0.6wt%, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 30ml/20*20cm 2, the operating time is less than 5min; Put into cryogenic refrigerator ,-40 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 10h makes without glue composite sponge dressing;
Prepared casting product is double-deck in conjunction with firm, see accompanying drawing, hybrid layer thickness 60-100 μ m, the about 250-300 μ of the spongy functional layer thickness in upper strata m, quality is comparatively soft, flexible, absorption speed is fast, after inhaling a small amount of water, spongy layer skeleton is not out of shape, and dressing overall air permeability, water absorption and comfortableness are good, and anti-microbial property is strong.
Embodiment 6
By 20*20cm 2100g/ m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 40ml RO pure water, be placed in ultra cold storage freezer ,-40 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 2.5wt%, take macromole chitosan 1.3g, micromolecule chitosan 0.2g, macromolecular collagen protein 0.8g, micromolecular collagen 0.2g, be dissolved in the aqueous acetic acid of 100ml 0.6wt%, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 40ml/20*20cm 2, the operating time is less than 5min; Put into cryogenic refrigerator ,-40 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 10-12h makes without glue composite sponge dressing;
Prepared casting product is double-deck in conjunction with firm, hybrid layer thickness 60-100 μ m, and the about 400-450 μ of the spongy functional layer thickness in upper strata m, quality softness, flexible, absorption speed is fast; Dressing overall air permeability, water absorption and comfortableness are good, and anti-microbial property is strong.
Embodiment 7
By 20*20cm 2100g/ m 2chitin fiber needle punched non-woven fabrics be laid in rustless steel refrigerator tray, at nonwoven surface spray 40ml RO pure water, be placed in ultra cold storage freezer ,-30 DEG C of precooling 30min; Preparation spongy layer material solution, total concentration 2.5wt%, take macromole chitosan 0.8g, micromolecule chitosan 0.2g, macromolecular collagen protein 0.8g, micromolecular collagen 0.2g, be dissolved in the aqueous acetic acid of 80ml 0.5wt%, at room temperature refiner is stirred to uniform state, ultrasonic degas, 20 DEG C of preservations; Take out the chitosan non-woven fabrics refrigerator tray through pre-freeze, rapidly spongy layer material solution is evenly sprayed on non-woven fabrics to spray flux 20ml/20*20cm 2, the operating time is less than 5min; Put into cryogenic refrigerator ,-50 DEG C of freezing 60min, proceed in freezer dryer fast again, and vacuum drying 11h makes without glue composite sponge dressing;
Prepared casting product is double-deck in conjunction with firm, hybrid layer thickness 60-100 μ m; The about 100-150 μ of the spongy functional layer thickness in upper strata m, quality is comparatively soft, and flexible, absorption speed is fast; Dressing overall air permeability, water absorption and comfortableness are good.
Application Example 1
By double-deck dressing prepared embodiment 6, for the experiment of pig back deep second degree burn, control sample is silver sulfadiazine/gauze and chitin fiber adhesive-bonded fabric dressing.Experimental result shows, for deep second degree burn, use the wound healing rate of experimental group of the prepared double-deck dressing of the present invention all higher than silver sulfadiazine/gauze matched group and chitin fiber adhesive-bonded fabric dressing matched group, the healing rate of double-deck dressing group reaches 92.5%, and matched group is all lower than 85.0%; While changing dressings, double-deck dressing and wound adhesion, and chitin fiber adhesive-bonded fabric dressing is changed dressings and is all sticked together ninety-nine times out of a hundred.This shows, double-deck dressing of the present invention is conducive to the healing of deep second degree burn wound, is suitable for wound dressing purposes.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

Claims (6)

1. a two-layer compound biological dressing, it is characterized in that: by chitin fiber non-woven fabrics and spongy layer material solution, under the condition that does not add adhesive, low-temperature freeze drying is chimeric forms in described dressing, lower floor is chitin fiber nonwoven layer, and upper strata is spongy functional layer; In described spongy layer material solution, contain macromole chitosan, micromolecule chitosan, macromolecular collagen protein, micromolecular collagen.
2. the preparation technology of two-layer compound biological dressing according to claim 1, it is characterized in that: in described spongy layer material solution, the quality proportioning of macromole chitosan, micromolecule chitosan, macromolecular collagen protein, micromolecular collagen is: 0.7-1.3:0.1-0.3:0.7-1.3:0.1-0.3, total concentration is 2.0-3.5wt%.
3. the preparation technology of two-layer compound biological dressing according to claim 2, it is characterized in that: described macromole chitosan molecule amount is greater than 50,000 Da, micromolecule chitosan mean molecule quantity 2000Da, macromolecular collagen protein molecular weight is greater than 80,000 Da, micromolecular collagen mean molecule quantity 1000Da.
4. two-layer compound biological dressing according to claim 1, is characterized in that: the chimeric thickness between the upper and lower is 30 ~ 120 μ m.
5. a technique of preparing two-layer compound biological dressing as claimed in claim 1, is characterized in that: comprise the following steps:
(1) after chitin fiber nonwoven surface spray pure water, freeze at-20 ~-40 DEG C;
(2) preparation spongy layer material solution, after stirring, ultrasonic degas;
(3) the chitin fiber non-woven fabrics of taking-up prefreezing, is evenly sprayed at spongy layer material solution on non-woven fabrics rapidly;
(4) by the chitin fiber non-woven fabrics that is coated with spongy layer material solution-40 ~-50 DEG C freezing after, vacuum drying makes two-layer compound biological dressing.
6. the preparation technology of two-layer compound biological dressing according to claim 5, is characterized in that: in step (3), the spray flux of spongy layer material solution is 20-60ml/20*20cm 2.
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CN105983131A (en) * 2015-02-13 2016-10-05 陕西巨子生物技术有限公司 Hydrogel collagen dressing especially suitable for oily skins
CN106309150A (en) * 2015-06-29 2017-01-11 山东国际生物科技园发展有限公司 Medical collagen dressing, and preparation method and applications thereof
CN105126150B (en) * 2015-09-26 2018-05-01 谈伟强 A kind of bandage of chitosan-containing mixture and preparation method thereof
CN105126150A (en) * 2015-09-26 2015-12-09 谈伟强 Adhesive bandage containing chitosan mixture and preparation method of adhesive bandage
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CN105126151A (en) * 2015-09-30 2015-12-09 陕西艾尔肤组织工程有限公司 Collagen dressing plaster and preparing method and application thereof
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