CN105088779A - Collagen attachment membrane and preparation method thereof - Google Patents

Collagen attachment membrane and preparation method thereof Download PDF

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CN105088779A
CN105088779A CN201510473858.4A CN201510473858A CN105088779A CN 105088779 A CN105088779 A CN 105088779A CN 201510473858 A CN201510473858 A CN 201510473858A CN 105088779 A CN105088779 A CN 105088779A
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polyvinyl alcohol
silk protein
spider silk
recombinant spider
composite fiber
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CN105088779B (en
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陈海佳
王一飞
葛啸虎
李丽娟
舒辉萍
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Guangzhou Saliai Biological Gene Engineering Co Ltd
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Guangzhou Saliai Biological Gene Engineering Co Ltd
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Abstract

The invention belongs to the field of medical cosmetology, and particularly relates to a collagen attachment membrane and a preparation method thereof. The collagen attachment membrane is manufactured by soaking an RGD recombinant spider silk protein-polyvinyl alcohol composite fiber membrane in a collagen solution containing alkaline fibroblast growth factors, and sequentially conducting drying and cross-linking and solidifying. The RGD recombinant spider silk protein-polyvinyl alcohol composite fiber membrane is modified through collagen and the alkaline fibroblast growth factors, promotion of the composite fiber membrane on wound surface cell growth is enhanced, and therefore the collagen attachment membrane can have a high wound surface healing rate. The experiment results show that after the wound surface is covered with the collagen attachment membrane for one week, the healing rate of the wound surface is larger than 22%; after the wound surface is covered with the collagen attachment membrane for two weeks, the healing rate of the wound surface is larger than 83%; after the wound surface is covered with the collagen attachment membrane for 4 weeks, the healing rate of the wound surface is larger than 99.9%.

Description

A kind of collagem membrane pastes and preparation method thereof
Technical field
The invention belongs to medical cosmetology field, particularly relate to a kind of collagem membrane and paste and preparation method thereof.
Background technology
Skin is the maximum organ of human body, is the natural cover for defense of human body, to maintaining the stable of vivo environment and stoping microorganism invasion to play an important role.In recent years, various burned patient number remains high every year clinically.Meanwhile, increasing sharply of global aged's ratio, the large area skin ulcers caused because of diseases such as diabetes also gets more and more.Therefore social extensive concern is subject to for the artificial skin of reparing skin defect, the investigation and application of Wound dressing.
Wound dressing requires higher to the biocompatibility of raw material and biologically active, and the material be applicable to is few.Spider's thread protein as high performance biomaterial, such as artificial tendon, artificial ligament and the organizational project such as artificial organs, tissue repair field of new have very tempting application prospect.But take up an area for jail, internecine nature because spider has, cultivation difficulty, can not large-scale cultivation, and the product silk amount of spider is little, and extraction process is very complicated again, and technical difficulty is large, therefore wants to utilize the natural spider's thread to be unpractical simultaneously.In recent years, the scientist of countries in the world has carried out a series of research, with the spider's thread protein utilizing gene technology to develop Prof. Du Yucang to the chemical composition of spider's thread protein matter, structure and spider's thread protein plasmagene composition.
RGD recombinant spider silk protein is a kind of spider's thread protein of Prof. Du Yucang.Existing correlative study at present utilizes electrostatic spinning technique, with RGD recombinant spider silk protein for raw material, itself and polyvinyl alcohol are carried out co-blended spinning, has prepared RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, and this composite cellulosic membrane is applied to Wound dressing field.But when this composite cellulosic membrane uses as Wound dressing, the healing effect of the surface of a wound is poor.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of collagem membrane to paste and preparation method thereof, when collagem membrane provided by the invention pastes and uses as Wound dressing, the healing effect of the surface of a wound is better.
The invention provides a kind of collagem membrane to paste, make through super-dry and crosslinking curing successively after being flooded in containing the collagen solution of basic fibroblast growth factor by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
Preferably, the porosity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 75 ~ 90%.
Preferably, the hydroscopicity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 3 ~ 5g/g.
Preferably, the swelling ratio of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 2 ~ 3g/g.
Preferably, the content of described basic fibroblast growth factor in described collagen solution is 100 ~ 2000IU/mL.
Preferably, the content of described collagen in described collagen solution is 1 ~ 20wt%.
The invention provides the preparation method that a kind of collagem membrane pastes, comprise the following steps:
A), by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film immersion containing in the collagen solution of basic fibroblast growth factor, the composite cellulosic membrane being impregnated with basic fibroblast growth factor and collagen solution is obtained;
B), the composite cellulosic membrane that a) obtains of step successively through super-dry and crosslinking curing, obtain collagem membrane and paste.
Preferably, the temperature of described dipping is 30 ~ 40 DEG C; The time of described dipping is 20 ~ 60min.
Preferably, the process of described crosslinking curing is specially:
Dried described composite cellulosic membrane carries out crosslinking curing in cross-linking agent solution;
The time of described crosslinking curing is 3 ~ 10h.
Preferably, described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is prepared by the following method:
1), RGD recombinant spider silk protein, polyvinyl alcohol and solvent, obtain spinning solution;
2), described spinning solution carries out spinning, obtains RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
Compared with prior art, the invention provides a kind of collagem membrane to paste and preparation method thereof.Collagem membrane provided by the invention is made through super-dry and crosslinking curing after pasting and being flooded in containing the collagen solution of basic fibroblast growth factor by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film successively.The present invention carries out modification by adopting collagen and basic fibroblast growth factor to RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, enhance the promotion of composite cellulosic membrane to surface of a wound Growth of Cells, thus make collagem membrane provided by the invention subsides show higher Wound healing rate.Experimental result shows, pasted by collagem membrane provided by the invention and cover the surface of a wound after 1 week, the healing rate of the surface of a wound is greater than 22%; Flap coverage is after 2 weeks, and the healing rate of the surface of a wound is greater than 83%; Flap coverage is after 4 weeks, and the healing rate of the surface of a wound is greater than 99.9%.
Detailed description of the invention
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The invention provides a kind of collagem membrane to paste, make through super-dry and crosslinking curing successively after being flooded in containing the collagen solution of basic fibroblast growth factor by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
Collagem membrane provided by the invention is made through super-dry and crosslinking curing after pasting and being flooded in containing the collagen solution of basic fibroblast growth factor by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film successively.In an embodiment provided by the invention, the thickness that described collagem membrane pastes is 0.01 ~ 0.2mm, and in another embodiment provided by the invention, the thickness that described collagem membrane pastes is 0.05 ~ 0.1mm.
In the present invention, described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is the framework material that collagem membrane provided by the invention pastes.In an embodiment provided by the invention, the porosity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 75 ~ 90%; In another embodiment provided by the invention, the porosity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 80 ~ 85%; In other embodiments provided by the invention, the porosity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 80.72 ~ 84.89%.In an embodiment provided by the invention, the hydroscopicity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 3 ~ 5g/g; In another embodiment provided by the invention, the hydroscopicity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 3.5 ~ 4.5g/g; In other embodiments provided by the invention, the hydroscopicity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 3.981 ~ 4.337g/g.In an embodiment provided by the invention, the swelling ratio of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 2 ~ 3g/g; In another embodiment provided by the invention, the swelling ratio of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 2.2 ~ 2.6g/g; In other embodiments provided by the invention, the swelling ratio of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 2.21 ~ 2.526g/g.In an embodiment provided by the invention, the diffusate of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film between fiber and intrastitial distributions ratios be 5 ~ 7g/g; In another embodiment provided by the invention, the diffusate of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film between fiber and intrastitial distributions ratios be 5.5 ~ 6.5g/g; In other embodiments provided by the invention, the diffusate of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film between fiber and intrastitial distributions ratios be 5.647 ~ 6.023g/g.
The source of the present invention to described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is not particularly limited, and can be commercially available, also can prepare in accordance with the following methods:
1), RGD recombinant spider silk protein, polyvinyl alcohol and solvent, obtain spinning solution;
2), described spinning solution carries out spinning, obtains RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
In the preparation method of RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film provided by the invention, first by RGD recombinant spider silk protein, polyvinyl alcohol and solvent.Wherein, described RGD recombinant spider silk protein is preferably provided by Ji'nan University; The average degree of polymerization of described polyvinyl alcohol is preferably 2400 ~ 2500; Described average degree of polymerization is that the model of the polyvinyl alcohol of 2400 ~ 2500 is preferably PVA-124; Described solvent is preferably formic acid.After RGD recombinant spider silk protein, polyvinyl alcohol and solvent are even, obtain spinning solution.In described spinning solution, the content of RGD recombinant spider silk protein is preferably 5 ~ 15wt%; In described spinning solution, the content of polyvinyl alcohol is preferably 5 ~ 15wt%.
Then obtained spinning solution is carried out spinning.The mode of described spinning is preferably electrostatic spinning.The specification of syringe that described electrostatic spinning adopts is preferably 2mL syringe; The voltage strength of described electrostatic spinning is preferably 60 ~ 80kV, is more preferably 80 ~ 85kV; The spinning solution flow of described electrostatic spinning is preferably 2 ~ 10mL/h, is more preferably 5 ~ 6mL/h; The distance that the syringe needle of described electrostatic spinning collects screen surfaces to spinning is preferably 10 ~ 30cm, is more preferably 20 ~ 25cm.The temperature of described electrostatic spinning is preferably 40 ~ 55 DEG C, is more preferably 45 ~ 50 DEG C.After spinning terminates, obtain RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
Collagem membrane provided by the invention pastes the RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film adopted and has good mechanical performance, non-stimulated to skin, there are higher porosity and good connected pore channel, well can wrap up the Porcine HGF of interpolation, this composite cellulosic membrane good permeability, is convenient to the derivation of wound fluid simultaneously.
In the present invention, the described collagen solution containing basic fibroblast growth factor is the solution be mixed to get by collagen, basic fibroblast growth factor (bFGF) and water.The source of the present invention to described collagen and basic fibroblast growth factor is not particularly limited, and adopts commercial goods.Wherein, described collagen is preferably provided by bio tech ltd of Xu Er U.S., Shijiazhuang; Described basic fibroblast growth factor is preferably provided by Ke Xin bio tech ltd, Beijing.In an embodiment provided by the invention, the content of described basic fibroblast growth factor in described collagen solution is 100 ~ 2000IU/mL; In another embodiment provided by the invention, the content of described basic fibroblast growth factor in described collagen solution is 500 ~ 1000IU/mL.In an embodiment provided by the invention, the content of described collagen in described collagen solution is 1 ~ 20wt%; In another embodiment provided by the invention, the content of described collagen in described collagen solution is 2 ~ 10wt%.In the present invention, by collagen and basic fibroblast growth factor, modification is carried out to RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, enhance the promotion of composite cellulosic membrane to surface of a wound Growth of Cells, thus make collagem membrane provided by the invention subsides show higher Wound healing rate.
The present invention carries out modification by adopting collagen and basic fibroblast growth factor to RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, enhance the promotion of composite cellulosic membrane to surface of a wound Growth of Cells, thus make collagem membrane provided by the invention subsides show higher Wound healing rate.Experimental result shows, pasted by collagem membrane provided by the invention and cover the surface of a wound after 1 week, the healing rate of the surface of a wound is greater than 22%; Flap coverage is after 2 weeks, and the healing rate of the surface of a wound is greater than 83%; Flap coverage is after 4 weeks, and the healing rate of the surface of a wound is greater than 99.9%.
The invention provides the preparation method that a kind of collagem membrane pastes, comprise the following steps:
A), by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film immersion containing in the collagen solution of basic fibroblast growth factor, the composite cellulosic membrane being impregnated with basic fibroblast growth factor and collagen solution is obtained;
B), the composite cellulosic membrane that a) obtains of step successively through super-dry and crosslinking curing, obtain collagem membrane and paste.
In preparation method provided by the invention, first by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film immersion containing in the collagen solution of basic fibroblast growth factor.Wherein, the porosity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is preferably 75 ~ 90%, is more preferably 80 ~ 85%, most preferably is 80.72 ~ 84.89%; The hydroscopicity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is preferably 3 ~ 5g/g, is more preferably 3.5 ~ 4.5g/g, most preferably is 3.981 ~ 4.337g/g.The swelling ratio of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is preferably 2 ~ 3g/g, is more preferably 2.2 ~ 2.6g/g, most preferably is 2.21 ~ 2.526g/g.The diffusate of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is preferably 5 ~ 7g/g with intrastitial distributions ratios between fiber, is more preferably 5.5 ~ 6.5g/g, most preferably is 5.647 ~ 6.023g/g.
In the present invention, the source of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is not particularly limited, can be commercially available, also can prepare in accordance with the following methods:
1), RGD recombinant spider silk protein, polyvinyl alcohol and solvent, obtain spinning solution;
2), described spinning solution carries out spinning, obtains RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
In the preparation method of RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film provided by the invention, first by RGD recombinant spider silk protein, polyvinyl alcohol and solvent.Described solvent is preferably formic acid.After RGD recombinant spider silk protein, polyvinyl alcohol and solvent are even, obtain spinning solution.In described spinning solution, the content of RGD recombinant spider silk protein is preferably 5 ~ 15wt%; In described spinning solution, the content of polyvinyl alcohol is preferably 5 ~ 15wt%.
Then, obtained spinning solution is carried out spinning.The mode of described spinning is preferably electrostatic spinning.The specification of syringe that described electrostatic spinning adopts is preferably 2mL syringe; The voltage strength of described electrostatic spinning is preferably 60 ~ 80kV, is more preferably 80 ~ 85kV; The spinning solution flow of described electrostatic spinning is preferably 2 ~ 10mL/h, is more preferably 5 ~ 6mL/h; The distance that the syringe needle of described electrostatic spinning collects screen surfaces to spinning is preferably 10 ~ 30cm, is more preferably 20 ~ 25cm.The temperature of described electrostatic spinning is preferably 40 ~ 55 DEG C, is more preferably 45 ~ 50 DEG C.After spinning terminates, obtain RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
In the present invention, the described collagen solution containing basic fibroblast growth factor is the solution be mixed to get by collagen, basic fibroblast growth factor (bFGF) and water.The content of described basic fibroblast growth factor in described collagen solution is preferably 100 ~ 2000IU/mL, is more preferably 500 ~ 1000IU/mL; The content of described collagen in described collagen solution is preferably 1 ~ 20wt%, is more preferably 2 ~ 10wt%.
In the present invention, described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is preferably 30 ~ 40 DEG C in the described temperature of flooding in the collagen solution of basic fibroblast growth factor that contains, and is more preferably 37 ~ 40 DEG C; The time of described dipping is preferably 20 ~ 60min, is more preferably 30 ~ 40min.
After dipping, obtain the composite cellulosic membrane being impregnated with basic fibroblast growth factor and collagen solution, described composite cellulosic membrane carries out drying.The mode of described drying is preferably natural air drying; The time of described drying is preferably 1 ~ 12h, is more preferably 2 ~ 5h.In the present invention, the composite cellulosic membrane being impregnated with basic fibroblast growth factor and collagen solution preferably carries out drying in a mold, thus makes dried composite cellulosic membrane have given shape and size.
After drying, dried composite cellulosic membrane carries out crosslinking curing.The process of described crosslinking curing is specially: dried described composite cellulosic membrane carries out crosslinking curing in cross-linking agent solution.Wherein, described crosslinking agent is preferably 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl); In described cross-linking agent solution, the content concn of crosslinking agent is preferably 0.01 ~ 0.05mol/L, is more preferably 0.03mol/L; The time of described crosslinking curing is preferably 5 ~ 12h, is more preferably 8 ~ 9h.After crosslinking curing terminates, obtain collagem membrane and paste.
After obtained collagem membrane pastes, collagem membrane is placed with in aseptic package bag, sterilizing, preserves.
Preparation method provided by the invention carries out modification by adopting collagen and basic fibroblast growth factor to RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, enhance the promotion of composite cellulosic membrane to surface of a wound Growth of Cells, thus the collagem membrane that the present invention is obtained subsides show higher Wound healing rate.Experimental result shows, pasted by collagem membrane provided by the invention and cover the surface of a wound after 1 week, the healing rate of the surface of a wound is greater than 22%; Flap coverage is after 2 weeks, and the healing rate of the surface of a wound is greater than 83%; Flap coverage is after 4 weeks, and the healing rate of the surface of a wound is greater than 99.9%.
For the purpose of clearer, be described in detail below by following examples.
Embodiment 1
1) RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is prepared
RGD recombinant spider silk protein (Ji'nan University provides) and polyvinyl alcohol (average degree of polymerization is 2400 ~ 2500) are dissolved in the formic acid solution of 98wt%, are mixed with the spinning solution containing 5wt%RGD recombinant spider silk protein and 5wt% polyvinyl alcohol.
Spinning solution is sucked in the syringe of 2mL specification, utilize electrostatic textile technology, under 80kV (voltage strength), 20cm (syringe needle is apart from collecting screen surfaces), 5ml/h (spinning solution flow), 45 DEG C of conditions, spinning solution is prepared into RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
The physico-chemical parameter of above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is detected, specific as follows:
The mensuration of porosity:
Under constant temperature, in 5ml specific gravity bottle, fill absolute ethyl alcohol, weigh (W1), and weigh composite cellulosic membrane (Ws).And film is placed in specific gravity bottle, leave standstill 15min, utilize vavuum pump to carry out degassed, until bubble-free effusion, ethanol is fully diffused in the hole of film, fills it up with ethanol, weigh (W2).The sample of full for leaching ethanol is taken out, (W3) is weighed to remaining ethanol and specific gravity bottle.Counting Formula: porosity=(W2-W3-Ws)/(W1-W3), show that the porosity of composite cellulosic membrane is 82.65%;
Hygroscopicity measures:
The preparation of solution A: 16.6gNaCl, 0.554gCaCl 2, use ddH 2o is settled to 200ml in volumetric flask, is diluted 10 times during use.
Under constant temperature at 37 DEG C, measure the dry weight (W) of composite cellulosic membrane, take the solution A of 40 times of film weight, film is placed in solution A, place 30min at 37 DEG C, film is taken out vertical hanging 1min, measure weight in wet base (W1), computing formula: the hygroscopicity of Unit Weight film is (W1-W)/W (unit: g/g), and the hydroscopicity measuring this composite cellulosic membrane is 3.981g/g;
Diffusate is between fiber and intrastitial distribution is compared and the mensuration of swelling ratio:
The preparation of solution A: 16.6gNaCl, 0.554gCaCl 2, use ddH 2o is settled to 200ml in volumetric flask, is diluted 10 times during use.
Under constant temperature at 37 DEG C, measure the dry weight (W) of composite cellulosic membrane, take the solution A of 40 times of film weight, the heavy solution A of 40 times of films is taken with beaker, film is divided into impartial two parts, is placed in solution A, at 37 DEG C, places 30min, film is taken out vertical hanging 30s, measure weight in wet base (W1); Be placed in a 50ml centrifuge tube, filter paper is covered with bottom centrifuge tube, to absorb the centrifugal moisture content sloughed from fiber, 400g, 20 DEG C, centrifugal 20min, measures the weight (W2) of dehydration caudacoria, film after centrifugal dehydration is dried to constant weight at 70 DEG C, weighs film dry weight (W3).Computing formula, diffusate is between fiber and intrastitial distribution ratio=(W1-W2)/(W2-W3), and showing that distribution is compared is 5.647g/g; Swelling ratio=(the W1-W2)/W3 of film, the swelling ratio obtaining membrane is 2.21g/g.
Carry out security test to above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, detailed process is:
Take rat as tested object, lose hair or feathers at its back, area is 3cm 2, obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is got 6cm 2immerse in 2ml physiological saline, 37 DEG C of constant temperature place 24h, with the formalin of 5wt% for positive controls, physiological saline is negative control group, RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film leaching liquor is experimental group, by the filter paper patch of each experimental group, cover with 4 layers of gauze of 20mm × 20mm, wrapping is fixing.After 4h, removing patch thing, mark patch position, blots after cleaning with warm water or 70% ethanol.After removing patch thing, 24h, 48h and 72h continuous observation draws the following conclusions table 1:
Table 1RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is to the excitant score result of skin
Can be found out by table 1, RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film that the present embodiment obtains is non-stimulated to skin.
2) bFGF-collagen solution is prepared
Collagen (bio tech ltd of Xu Er U.S., Shijiazhuang provides) is dissolved in sterilized water the collagen solution being mixed with 2wt%, add bFGF (Ke Xin bio tech ltd, Beijing provides) by 500IU/ml, obtain bFGF-collagen solution.
3) prepare collagem membrane to paste
Above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film constant temperature 37 DEG C is immersed in 30min in above-mentioned obtained bFGF-collagen solution, then puts into gel mold room temperature nature gel drying 2h, obtain the composite cellulosic membrane of drying and moulding.
The composite cellulosic membrane of drying and moulding is put into the crosslinked 8h reinforcing of EDCHCl solution that concentration is 0.01 ~ 0.05mol/L, obtain the collagem membrane subsides that thickness is 0.05mm.
Finally obtained collagem membrane is placed with into aseptic package bag, carries out ethylene oxide sterilizing.
Embodiment 2
1) RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is prepared
RGD recombinant spider silk protein (Ji'nan University provides) and polyvinyl alcohol (average degree of polymerization is 2400 ~ 2500) are dissolved in the formic acid solution of 98wt%, are mixed with the spinning solution containing 7.5wt%RGD recombinant spider silk protein and 7.5wt% polyvinyl alcohol.
Spinning solution is sucked in the syringe of 2mL specification, utilize electrostatic textile technology, under 80kV (voltage strength), 20cm (syringe needle is apart from collecting screen surfaces), 5ml/h (spinning solution flow), 45 DEG C of conditions, spinning solution is prepared into RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
Detect according to the method in embodiment 1 physico-chemical parameter of above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, result is: voidage 84.89%, hydroscopicity 4.337g/g, diffusate between fiber and intrastitial distribution be 2.526g/g than 6.023g/g, swelling ratio.
2) bFGF-collagen solution is prepared
Collagen (bio tech ltd of Xu Er U.S., Shijiazhuang provides) is dissolved in sterilized water the collagen solution being mixed with 5wt%, add bFGF (Ke Xin bio tech ltd, Beijing provides) by 750IU/ml, obtain bFGF-collagen solution.
3) prepare collagem membrane to paste
Above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film constant temperature 37 DEG C is immersed in 30min in above-mentioned obtained bFGF-collagen solution, then puts into gel mold room temperature nature gel drying 2h, obtain the composite cellulosic membrane of drying and moulding.
The composite cellulosic membrane of drying and moulding is put into the crosslinked 8h reinforcing of EDCHCl solution that concentration is 0.03mol/L, obtain the collagem membrane subsides that thickness is 0.1mm.
Finally obtained collagem membrane is placed with into aseptic package bag, carries out ethylene oxide sterilizing.
Embodiment 3
1) RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is prepared
RGD recombinant spider silk protein (Ji'nan University provides) and polyvinyl alcohol (average degree of polymerization is 2400 ~ 2500) are dissolved in the formic acid solution of 98wt%, are mixed with the spinning solution containing 15wt%RGD recombinant spider silk protein and 15wt% polyvinyl alcohol.
Spinning solution is sucked in the syringe of 2mL specification, utilize electrostatic textile technology, under 80kV (voltage strength), 20cm (syringe needle is apart from collecting screen surfaces), 5ml/h (spinning solution flow), 45 DEG C of conditions, spinning solution is prepared into RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
Detect according to the method for embodiment 1 physico-chemical parameter of above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film, result is: voidage 80.72%, hydroscopicity 4.165g/g, diffusate between fiber and intrastitial distribution be 2.314g/g than 5.871g/g, swelling ratio.
2) bFGF-collagen solution is prepared
Collagen (bio tech ltd of Xu Er U.S., Shijiazhuang provides) is dissolved in sterilized water the collagen solution being mixed with 10wt%, add bFGF (Ke Xin bio tech ltd, Beijing provides) by 1000IU/ml, obtain bFGF-collagen solution.
3) prepare collagem membrane to paste
Above-mentioned obtained RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film constant temperature 37 DEG C is immersed in 30min in above-mentioned obtained bFGF-collagen solution, then puts into gel mold room temperature nature gel drying 2h, obtain the composite cellulosic membrane of drying and moulding.
The composite cellulosic membrane of drying and moulding is put into the crosslinked 8h reinforcing of EDCHCl solution that concentration is 0.05mol/L, obtain the collagem membrane subsides that thickness is 0.1mm.
Finally obtained collagem membrane is placed with into aseptic package bag, carries out ethylene oxide sterilizing.
Embodiment 4
Collagem membrane is attached to the test of the surface of a wound
Test with 100 new zealand rabbits, set up wounds in animals model, be divided into 5 groups at random: embodiment 1 group, embodiment 2 groups, embodiment 3 groups, comparative example group and blank group, often organize 20.Wherein, embodiment 1 group uses the obtained collagem membrane of embodiment 1 to paste; Embodiment 2 groups uses the obtained collagem membrane of embodiment 2 to paste; The collagem membrane subsides that embodiment 3 groups of embodiments 3 are obtained; RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film that comparative example group uses embodiment 1 obtained.
After new zealand rabbit anesthesia sterilization, manufacture the otch of 2cm × 2cm, complete removal skin holostrome, paste flap coverage with the embodiment 1 group of surface of a wound formed objects, embodiment 2 groups, embodiment 3 groups and comparative example group film respectively, the blank surface of a wound does not do special treatment, then sterile pressurized wrapping.Postoperative intramuscular injection penicillin 10u/kgbw, every day 2 times, injects for three days on end, and prevention is infected.Change dressings weekly, remove each group of film and paste, by former method wrapping after wound clean.Observe the change of each treated animal surface of a wound after 1 to 4 week, calculate Wound healing rate, Wound healing rate=(original surface of a wound area-existing surface of a wound area)/original surface of a wound area × 100%, result is as following table 2:
The Wound healing rate that the postoperative different time points of table 2 is respectively organized
Group First week Second week 4th week
Blank 15.91+3.42 65.13+2.74 99.94+0.05
Comparative example 17.02+3.33 70.34+2.53 99.95+0.02
Embodiment 1 23.76+1.80 83.58+0.01 99.97+0.03
Embodiment 2 25.16+1.40 85.73+0.01 99.99+0.01
Embodiment 3 22.82+1.51 83.05+0.01 99.96+0.04
As shown in Table 2, in the 2nd week, embodiment group Wound healing rate is much larger than other group.From the observation of continuous surrounding, the 1st Wednesday group all there is congestion and edema, embodiment group is the slightest, and the collagem membrane of embodiment group pastes thinning and transparent, all sees that granulation tissue is formed bottom the surface of a wound, oozes out and lacks; 2nd week embodiment group is formed face and is obviously reduced, without obvious inflammatory exudate, and comparative example group and blank to form face obviously red and swollen, have inflammatory exudate; 4th week blank forms face has comparatively zonule to be epithelialization, and other form the complete protective epithelium in face, but the surface of a wound of comparative example group has clear scar contracture, and embodiment is formed face and had no clear scar contracture.When can illustrate that the collagem membrane subsides that the embodiment of the present invention provides use as Wound dressing by the observation of continuous surrounding, the healing effect of the surface of a wound is better.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. a collagem membrane pastes, and through super-dry and crosslinking curing makes successively after being flooded in containing the collagen solution of basic fibroblast growth factor by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
2. collagem membrane according to claim 1 pastes, and it is characterized in that, the porosity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 75 ~ 90%.
3. collagem membrane according to claim 1 pastes, and it is characterized in that, the hydroscopicity of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 3 ~ 5g/g.
4. collagem membrane according to claim 1 pastes, and it is characterized in that, the swelling ratio of described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is 2 ~ 3g/g.
5. collagem membrane according to claim 1 pastes, and it is characterized in that, the content of described basic fibroblast growth factor in described collagen solution is 100 ~ 2000IU/mL.
6. collagem membrane according to claim 1 pastes, and it is characterized in that, the content of described collagen in described collagen solution is 1 ~ 20wt%.
7. a preparation method for collagem membrane subsides, comprises the following steps:
A), by RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film immersion containing in the collagen solution of basic fibroblast growth factor, the composite cellulosic membrane being impregnated with basic fibroblast growth factor and collagen solution is obtained;
B), the composite cellulosic membrane that a) obtains of step successively through super-dry and crosslinking curing, obtain collagem membrane and paste.
8. preparation method according to claim 7, is characterized in that, the temperature of described dipping is 30 ~ 40 DEG C; The time of described dipping is 20 ~ 60min.
9. preparation method according to claim 7, is characterized in that, the process of described crosslinking curing is specially:
Dried described composite cellulosic membrane carries out crosslinking curing in cross-linking agent solution;
The time of described crosslinking curing is 3 ~ 10h.
10. preparation method according to claim 7, is characterized in that, described RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film is prepared by the following method:
1), RGD recombinant spider silk protein, polyvinyl alcohol and solvent, obtain spinning solution;
2), described spinning solution carries out spinning, obtains RGD recombinant spider silk protein-polyvinyl alcohol composite fiber film.
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