CN103756005B - A kind of Composite collagen sponge and preparation method thereof - Google Patents
A kind of Composite collagen sponge and preparation method thereof Download PDFInfo
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- CN103756005B CN103756005B CN201410010048.0A CN201410010048A CN103756005B CN 103756005 B CN103756005 B CN 103756005B CN 201410010048 A CN201410010048 A CN 201410010048A CN 103756005 B CN103756005 B CN 103756005B
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Abstract
The invention discloses a kind of Composite collagen sponge and preparation method thereof, it is characterized in that: it is cross-linked by adding linking agent after a certain proportion of fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract lyophilize, through being washed to neutrality, secondary freeze drying and the soft spongy dressing of the porous made again; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 70-80:10-20:5-10.The present invention adopts collagen protein, Bletilla glucomannan and Root of Common Lamiophlomis to be main raw material, develops a kind of novel wound dressing, has no side effect, have good biocompatibility; There is hemostasis clinically, wound healing, stop effect that sepage is overflowed, tearing strength>=600g/cm
3, water-intake rate>=30 times; Have good water absorbability, and improve the degradation property of collagen sponge, and its preparation process being simple, cheap and easy to get, is a kind of bio-medical material preferably.
Description
Technical field
The invention belongs to field of biological product, be specifically related to a kind of Composite collagen sponge and preparation method thereof.
Background technology
The main composition of collagen sponge is collagen protein, and it is similar to human collagen protein structure, is well suited for reparation and the regeneration of human organ.Collagen protein can promote the growth of wound healing and granulation tissue, has good hemostasis and filling effect, can be used for wound healing, hemostasis, the filling of operation residual cavity etc.The impact of three-dimensional structure on cytoactive of timbering material and support should be considered in the process preparing porous support.Concerning one can promote cell adhesion and growth have bioactive support, good biocompatibility must be had, and certain degradable in vivo speed can be kept.Support should have suitable mean pore size, and ensure appropriate cell adhesion area while making cell carry out moving between hole, collagen sponge is acknowledged as a kind of suitable medical material.
At present, the collagen protein of domestic use is mainly from skin and the heel string of pig and ox, but the collagen product that the Mammals such as ox, pig is main source exists problems such as raw material sources are limited, manufacture a finished product height and the pathogeny such as mad cow disease, foot and mouth disease are propagated potentially dangerous; And due to the reason such as religion and custom, the collagen protein in pig source is restricted in the application of certain areas.Therefore, in the urgent need to seeking quality and the better collagen sources of security.The fishery products wastes such as the annual fish scale fish-skin of China about have more than 40 ten thousand tons, these fishery products wastes are all containing abundant collagen protein (as skin, bone, squama etc.), its amino acid composition and Lu Sheng mammal collagen no significant difference, and there is not the communicable disease of infecting both domestic animals and human in it, there is great development and utilization be worth, but these wastes are except small portion is used to production feed, and major part is but dropped.Therefore, that develops the fishery products wastes such as fish-skin utilizes new way, not only can improve the added value of processing of aquatic products, and can reduce environmental pollution, have good economic and social benefit.Collagen sponge is extensively approved as the three-dimensional rack building active artificial skin, but its vivo degradation is fast, snappiness is poor, degraded is large by the impact of local factor, degradation speed controllability is not as artificial polymer, bad mechanical property, is difficult in a liquid keep its pore texture, limits its application.
The preparation method of current fish source collagen albumen is a lot, comprise acid system, alkaline process, enzyme process, salt method, hot-water process etc., but these traditional methods all exists the shortcomings such as preparation time length, complex process.The denaturation temperature of fish source collagen albumen is at about 30 DEG C, and the too high collagen protein that causes of Extracting temperature loses biological activity, and collagen protein internal structure changes, function reduction, thus is difficult to produce a desired effect.
Italy product G ELFIX (Ossein sheet), was the lyophilized collagen taking from ox heel string, can promotes wound healing, as the Be very effective of surgical operation weighting agent and hemostatic drug, of many uses, in import China in 1992.But the Ossein sheet of import, expensive, be only collagen dressing, without anti-inflammatory and analgesic effect, obvious not as medicine carrying dressing to the short more property of wound.
Summary of the invention
The object of this invention is to provide a kind of Composite collagen sponge and preparation method thereof, shortcoming that the Composite collagen sponge prepared by the present invention can improve that collagen sponge is easily degraded etc., and overcome the deficiency of existing collagen sponge.
The present invention is achieved through the following technical solutions:
A kind of Composite collagen sponge, it is characterized in that: it is cross-linked by adding linking agent after a certain proportion of fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract lyophilize, through being washed to neutrality, secondary freeze drying and the soft spongy dressing of the porous made again; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 70-80:10-20:5-10.
Further, the preparation method of described Composite collagen sponge, is characterized in that comprising the following steps:
1) fish source collagen albumen being dissolved in volume fraction is in the acetic acid solution of 2%, makes the collagen solution that massfraction is 0.5%;
2) Bletilla glucomannan is immersed distilled water, after fully expanding, put in the water-bath of 50-60 DEG C and make it dissolve, be diluted with water to and make the Bletilla glucomannan solution that massfraction is 1-5%;
3) by above-mentioned obtained collagen solution and the mixing of Bletilla glucomannan solution, add Radix Lamiophlomidis Rotatae extract fine powder, stir;
4) by the mixing solutions moulding after mixing, obtain spongy material, spongy material is immersed linking agent and is cross-linked through lyophilize, cross-linking products is repeatedly extremely neutral through distilled water flushing; Glutaraldehyde or the concentration of described linking agent to be volume fraction be 0.2-0.3% are the EDC of 40-60mM, and crosslinking time is 12-24h;
5) being placed in vacuum drying oven drying by repeatedly rinsing to the spongy cross-linking products of neutral preparation, obtaining Composite collagen sponge, then after being cut into different size, oxirane disinfection, packaging, to obtain final product.
Preferably, the preparation method of described fish source collagen albumen is: following all operations all carries out under lower than the condition of 20 DEG C; The NaOH solution of the fish skin raw material 0.1mol/L after thawing is soaked 24h and removes foreign protein, subsequently with deionized water fully wash to neutrality and with 10% n-butanol solvent soak degreasing 24h, with deionized water repetitive scrubbing, drain after add moment transformation extract fill with on high-pressure impregnation tank in, add the acetic acid solution of appropriate 0.5mol/L, inflating pressure 8 ~ 10kgf/cm
2, soak 1-2 hour; Material in high-pressure impregnation tank is put into the low pressure extraction filling that moment transformation extraction is filled with, the acetic acid solution adding 0.5mol/L stirs and extracts 12h, repeat extraction 2 times, extract residue to continue to extract 24h with stirring containing 1.0% (W/V) pepsic 0.5mol/L acetic acid, merge No. 3 extracting solution centrifugations, obtain crude extract; In crude extract, adding NaCl to salt concn is 0.9mol/L, filter after leaving standstill the 24h that saltouts, use acetic acid and the distill water dialysis of 0.1mol/L after the acetic acid solution redissolution of collagen deposit 0.5mol/L successively, after last lyophilize, obtain fish source collagen protein sample;
The low pressure extraction filling comprising and be provided with agitator is filled with in described moment transformation extraction, and low pressure is extracted filling and is provided with the high-pressure impregnation tank connected by coupling cock, and described high-pressure impregnation tank top is provided with closed opening for feed, tensimeter and is connected the intake valve of air pump; Described low pressure is extracted the tank body of filling with and is provided with closed opening for feed, slag notch and baiting valve.
Preferably, the preparation method of described Bletilla glucomannan is: by the bletilla striata, and pulverize, add 50-60 DEG C of water soaking 6-8h of 6-8 times amount, stir constantly, filter, filtrate is for subsequent use; The 50-60 DEG C of water soaking 2 times of dregs of a decoction 4-6 times amount again, soaks 4h at every turn, filters; Merge 3 filtrates, be condensed into paste, add 95% ethanol after cooling, make alcohol content reach 70%-75%, stir, namely separate out a large amount of cotton-shaped heavy liquid, leave standstill 1h, vacuum filtration is to dry, and vacuum-drying, porphyrize, to obtain final product.
Preferably, the preparation method of described Radix Lamiophlomidis Rotatae extract: get the Root of Common Lamiophlomis medicinal material after pulverizing, with water or 40-70% extraction using alcohol 2-3 time, united extraction liquid, concentrated, by macroporous adsorbent resin on concentrated solution, with water and the removal of impurities of 10-30% ethanol elution, then use 40-80% ethanol elution, collect 40-80% ethanol eluate, concentrate drying, obtains Radix Lamiophlomidis Rotatae extract.
The bletilla striata is applied long-standing as convergence, hemostatic drug, and haemostatic effect is definitely reliable, and its mechanism of action is relevant with a large amount of Bletilla glucomannan contained by it.Bletilla striata quality sticks greasy, and containing colloid, it can improve local blood circulation, promotes epithelial cell reparation, can stop blooding, sore, moisturizing and myogenic.The white friendly glue extracted from the bletilla striata is made certain thickness film by experimentation on animals, spread on blutpunkte or face, fuse with the surface of a wound at once, be difficult to peel off, there is stronger bonding strength, and the one side of contact wound dissolves, discontiguous one side still maintains the original state, the effect of serve rapid hemostasis, protecting from infection.Bletilla striata extract is also demonstrated experimentally to have and shortens clotting time, suppression human body to cellulosic solvency action, and can form rapidly artificial thrombus, not only trauma hemostasis is commonly used, and also has good effect to visceral hemorrhage.The bletilla striata has significantly promotion keratinocyte effect, and this short migration effect has material impact to treatment skin wound, hemostasis and immediate union, further illustrates reparation and metabolic process that Bletilla glucomannan has participated in damaged tissue or cell directly.
Root of Common Lamiophlomis Lamiophlomisrotata (Benth.) Kudo system Labiatae acaulescence per nnial herb, with herb or root and root stock people medicine, is that the national folks such as Tibetan, illiteracy, Nahsi commonly use herbal medicine.There is hemostasis, analgesia detumescence, promoting blood circulation and removing blood stasis, the effect of mending marrow promoting the circulation of qi and reunion of fractured tendons and bones.Be loaded in Tibetan medicine's masterpiece Four-Volume Medical Code, " moon king medicine is examined " the earliest, there is promoting blood circulation and stopping pain, change the function of hemostasis of becoming silted up, clinically for the edge of a knife pain, hemorrhage of multiple surgical site infections, exogenous injury, muscles and bones is sprained, rheumatic arthralgia and uterine bleeding, dysmenorrhoea, tooth swelling and pain of the heel, hemorrhage etc.Its main active ingredient is flavonoid, iridoids and phenylethyl alcohol glycoside.
Relative to prior art, the invention has the beneficial effects as follows:
1) the present invention adopts collagen protein, Bletilla glucomannan and Root of Common Lamiophlomis to be main raw material, develop a kind of novel wound dressing, detect (acute toxicity test, irritation test, sensitivity test and hemolytic test) through toxicology to be all negative, have no side effect, there is good biocompatibility; Have hemostasis clinically, wound healing, the effect stoping sepage to be overflowed, has good clinical efficacy, and raw material sources enriches, cheap, possesses the prospect applied.Through test, the tearing strength>=600g/cm of Composite collagen sponge of the present invention
3(Ossein sheet>=500g/cm
3), water-intake rate>=30 times (Ossein sheet>=20 times), its water absorbability and tearing strength are significantly better than Ossein sheet.
2) combination of Bletilla glucomannan, Root of Common Lamiophlomis and collagen protein is except can promoting wound healing, also may strengthen its mechanical property; Bletilla glucomannan belongs to polyose, and collagen belongs to protein-based, and protein and sugar class may exist hydrogen bond action and electrostatic interaction together, adds crosslinked action, is cross-linked with each other between amido, hydroxyl etc., improves its mechanical strength further.Swelling property and snappiness that Bletilla glucomannan can increase collagen sponge as film forming material because its matter sticks, it is fast to the adhesion speed of the surface of a wound, adheres to fastness strong, good springiness, good permeability; Bletilla glucomannan has hemostatic and antibacterial and promotes wound healing, improves local blood circulation, can stop blooding, sore, moisturizing and myogenic; Root of Common Lamiophlomis has the effects such as analgesic hemostatic, promoting blood circulation and removing blood stasis, antisepsis and anti-inflammation.The present invention will have anastaltic Bletilla glucomannan and collagen protein and Root of Common Lamiophlomis compatibility that is promoting blood circulation and removing blood stasis, analgesic hemostatic, and one loose one receives, and has hemostasis and does not stay the wonderful of the stasis of blood, and can accelerate hemostatic effect.
3) the present invention adopts acid system and acid+Collagen Extraction with Enzyme albumen, raw material after pre-treatment is after high-pressure impregnation, water enters in cytolemma in a large number, then entering low pressure through abrupt release pressure extracts in filling, effectively can burst cytolemma, be conducive to the Fast Stripping of effective constituent, its method is easy and simple to handle, product yield high (being that raw material product yield reaches more than 45% in fish-skin).Unbound state uncrosslinked in raw material is after main collagen protein extracts by acid extraction method, residue uses acid+Enzymatic Extraction again, by pepsic hydrolytic action, dissociate and extract the collagen protein be under natural cross-linked state, the end peptide moiety of the non-helical structure at type i collagen two ends is excised, makes it to become solvable and don't affect the spirane structure of tropocollagen molecule; In whole extraction, purge process, the NaCl means of saltouing repeatedly are adopted to remove the collagenous portion of degraded or sex change, simultaneously because extraction environment temperature remains at less than 20 DEG C, the collagen protein biological activity of extraction is not destroyed, well ensure that the quality of collagen protein.Adopt temperature leaching+alcohol deposition method extraction purification Bletilla glucomannan, not only decrease foreign matter content, and technique simply, greatly reduce production cost, obtained Bletilla glucomannan purity is high.
4) the present invention is cross-linked after Bletilla glucomannan, Root of Common Lamiophlomis and the drying of fish source collagen protein frozen, its in the material residual is avoided through repeatedly washing removing linking agent, again carry out lyophilize, crosslinking process and secondary freeze drying technique not only make the physical strength of product greatly improve, good biocompatibility, and improving degradation rate and porosity, test shows that its porosity is more than 90%; The degradation rate of Composite collagen sponge lower than the degradation rate separating Er Feisi sheet, will overcome the feature that collagen is easily degraded to a certain extent.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the equipment that the present invention uses is described further:
Fig. 1 is the structural representation that moment pressure changeable extraction is filled with;
In figure: 1 be high-pressure impregnation tank, 2 be visor, 3 be closed opening for feed, 4 is tensimeter, 5 be the intake valve, 6 connecting air pump is low pressure extractor, 7 be agitator, 8 be coupling cock, 9 be slag notch, 10 be baiting valve.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.
Embodiment one: the preparation of Composite collagen sponge
1, the preparation of fish source collagen albumen: following all operations all carries out under lower than the condition of 20 DEG C; The NaOH solution of the fish skin raw material 0.1mol/L after thawing is soaked 24h and removes foreign protein, subsequently with deionized water fully wash to neutrality and with 10% n-butanol solvent soak degreasing 24h, with deionized water repetitive scrubbing, drain after add moment transformation extract fill with on high-pressure impregnation tank in, add the acetic acid solution (1:2 of 0.5mol/L, W/V), inflating pressure 8 ~ 10kgf/cm
2, soak 2 hours; Material in high-pressure impregnation tank is put into the low pressure extraction filling that moment transformation extraction is filled with, add the acetic acid solution (1:40 of 0.5mol/L, W/V) extraction 12h is stirred, repeat extraction 2 times, extracting residue continues with containing 1.0% (W/V) pepsic 0.5mol/L acetic acid (1:40, W/V) stir extraction 24h, merge No. 3 extracting solution centrifugations, obtain crude extract; In crude extract, adding NaCl to salt concn is 0.9mol/L, filter after leaving standstill the 24h that saltouts, use acetic acid and the distill water dialysis of 0.1mol/L after the acetic acid solution redissolution of collagen deposit 0.5mol/L successively, after last lyophilize, obtain fish source collagen protein sample.
As shown in Figure 1, the low pressure extraction filling 6 comprising and be provided with agitator 7 is filled with in described moment transformation extraction, low pressure is extracted filling 6 and is provided with the high-pressure impregnation tank 1 connected by coupling cock 8, and described high-pressure impregnation tank 1 top is provided with closed opening for feed 3, tensimeter 4 and is connected the intake valve 5 of air pump; The tank body that described low pressure extracts filling 6 is provided with closed opening for feed 3, slag notch 9 and baiting valve 10.
2, the preparation of Bletilla glucomannan: by the bletilla striata, pulverize, add 60 DEG C of water soaking 8h of 8 times amount, stir constantly, filter, filtrate is for subsequent use; The dregs of a decoction add 60 DEG C of water soakings 2 times of 6 times amount again, soak 4h at every turn, filter; Merge 3 filtrates, be condensed into paste, add 95% ethanol after cooling, make alcohol content reach 70%, stir, namely separate out a large amount of cotton-shaped heavy liquid, leave standstill 1h, vacuum filtration is to dry, and vacuum-drying, porphyrize, to obtain final product.
3, the preparation of Radix Lamiophlomidis Rotatae extract: get the Root of Common Lamiophlomis medicinal material after pulverizing, by 10 times amount water boiling and extraction 3 times, united extraction liquid, when to be concentrated into medicine liquid volume be 1.5 times amount of quality of medicinal material, by macroporous adsorbent resin DM130 on concentrated solution, with the water elution removal of impurities of 5 times of column volumes, again with 30% ethanol elution removal of impurities of 3 times of column volumes, then use 80% ethanol elution, collect 80% ethanol eluate, concentrate drying, obtains Radix Lamiophlomidis Rotatae extract.
4, the preparation of Composite collagen sponge:
1) above-mentioned fish source collagen albumen being dissolved in volume fraction is in the acetic acid solution of 2%, makes the collagen solution that massfraction is 0.5%;
2) above-mentioned Bletilla glucomannan is immersed distilled water, after fully expanding, put in the water-bath of 50-60 DEG C and make it dissolve, be diluted with water to and make the Bletilla glucomannan solution that massfraction is 1%;
3) by above-mentioned obtained collagen solution and the mixing of Bletilla glucomannan solution, then add Radix Lamiophlomidis Rotatae extract and stir; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 75:15:10;
4) by the mixing solutions moulding after mixing, obtain spongy material, spongy material is immersed linking agent and is cross-linked through lyophilize, cross-linking products is repeatedly extremely neutral through distilled water flushing; The glutaraldehyde of described linking agent to be volume fraction be 0.2-0.3%, crosslinking time is 12h;
5) being placed in vacuum drying oven drying by repeatedly rinsing to the spongy cross-linking products of neutral preparation, obtaining Composite collagen sponge, then after being cut into different size, oxirane disinfection, packaging, to obtain final product.
Embodiment two: the preparation of Composite collagen sponge
1, the preparation of fish source collagen albumen: following all operations all carries out under lower than the condition of 20 DEG C; The NaOH solution of the fish skin raw material 0.1mol/L after thawing is soaked 24h and removes foreign protein, subsequently with deionized water fully wash to neutrality and with 10% n-butanol solvent soak degreasing 24h, with deionized water repetitive scrubbing, drain after add moment transformation extract fill with on high-pressure impregnation tank in, add the acetic acid solution (1:1 of 0.5mol/L, W/V), inflating pressure 8 ~ 10kgf/cm
2, soak 1 hour; Material in high-pressure impregnation tank is put into the low pressure extraction filling that moment transformation extraction is filled with, add the acetic acid solution (1:40 of 0.5mol/L, W/V) extraction 12h is stirred, repeat extraction 2 times, extracting residue continues with containing 1.0% (W/V) pepsic 0.5mol/L acetic acid (1:40, W/V) stir extraction 24h, merge No. 3 extracting solution centrifugations, obtain crude extract; In crude extract, adding NaCl to salt concn is 0.9mol/L, filter after leaving standstill the 24h that saltouts, use acetic acid and the distill water dialysis of 0.1mol/L after the acetic acid solution redissolution of collagen deposit 0.5mol/L successively, after last lyophilize, obtain fish source collagen protein sample.
2, the preparation of Bletilla glucomannan: by the bletilla striata, pulverize, add 50 DEG C of water soaking 6h of 6 times amount, stir constantly, filter, filtrate is for subsequent use; The dregs of a decoction add 50 DEG C of water soakings 2 times of 4 times amount again, soak 4h at every turn, filter; Merge 3 filtrates, be condensed into paste, add 95% ethanol after cooling, make alcohol content reach 75%, stir, namely separate out a large amount of cotton-shaped heavy liquid, leave standstill 1h, vacuum filtration is to dry, and vacuum-drying, porphyrize, to obtain final product.
3, the preparation of Radix Lamiophlomidis Rotatae extract: get the Root of Common Lamiophlomis medicinal material after pulverizing, with 60% extraction using alcohol 2 times of 10 times amount volumes, united extraction liquid, when to be concentrated into relative density be 1.05, by macroporous adsorbent resin D101 on concentrated solution, with the water of 6 times amount column volumes and 10% ethanol elution removal of impurities, use 40% ethanol elution again, collect 40% ethanol eluate, concentrate drying, obtain Radix Lamiophlomidis Rotatae extract.
4, the preparation of Composite collagen sponge:
1) above-mentioned fish source collagen albumen being dissolved in volume fraction is in the acetic acid solution of 2%, makes the collagen solution that massfraction is 0.5%;
2) above-mentioned Bletilla glucomannan is immersed distilled water, after fully expanding, put in the water-bath of 50-60 DEG C and make it dissolve, be diluted with water to and make the Bletilla glucomannan solution that massfraction is 1-5%;
3) by above-mentioned obtained collagen solution and the mixing of Bletilla glucomannan solution, stir, remix adds Radix Lamiophlomidis Rotatae extract fine powder, stirs; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 80:10:5.
4) by the mixing solutions moulding after mixing, obtain spongy material, spongy material is immersed linking agent and is cross-linked through lyophilize, cross-linking products is repeatedly extremely neutral through distilled water flushing; The EDC of described linking agent to be concentration be 40-60mM, crosslinking time is 24h;
5) being placed in vacuum drying oven drying by repeatedly rinsing to the spongy cross-linking products of neutral preparation, obtaining Composite collagen sponge, then after being cut into different size, oxirane disinfection, packaging, to obtain final product.
Embodiment three: the preparation of Composite collagen sponge:
1) fish source collagen albumen (with embodiment two) being dissolved in volume fraction is in the acetic acid solution of 2%, makes the collagen solution that massfraction is 0.5%;
2) Bletilla glucomannan of embodiment one is immersed distilled water, after fully expanding, put in the water-bath of 50-60 DEG C and make it dissolve, be diluted with water to and make the Bletilla glucomannan solution that massfraction is 2%;
3) by above-mentioned obtained collagen solution and the mixing of Bletilla glucomannan solution, stir, remix adds Radix Lamiophlomidis Rotatae extract fine powder (with embodiment two), stirs; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 70:20:10.
4) by the mixing solutions moulding after mixing, obtain spongy material, spongy material is immersed linking agent and is cross-linked through lyophilize, cross-linking products is repeatedly extremely neutral through distilled water flushing; The EDC of described linking agent to be concentration be 40-60mM, crosslinking time is 24h;
5) being placed in vacuum drying oven drying by repeatedly rinsing to the spongy cross-linking products of neutral preparation, obtaining Composite collagen sponge, then after being cut into different size, oxirane disinfection, packaging, to obtain final product.
Embodiment four: the preparation of Composite collagen sponge:
1) fish source collagen albumen (purchased from French Luo Sailuo company, being fish scale collagen) being dissolved in volume fraction is in the acetic acid solution of 2%, makes the collagen solution that massfraction is 0.5%;
2) Bletilla glucomannan of embodiment one is immersed distilled water, after fully expanding, put in the water-bath of 50-60 DEG C and make it dissolve, be diluted with water to and make the Bletilla glucomannan solution that massfraction is 2%;
3) by above-mentioned obtained collagen solution and the mixing of Bletilla glucomannan solution, stir, remix adds Radix Lamiophlomidis Rotatae extract fine powder (with embodiment one), stirs; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 75:15:8.
4) by the mixing solutions moulding after mixing, obtain spongy material, spongy material is immersed linking agent and is cross-linked through lyophilize, cross-linking products is repeatedly extremely neutral through distilled water flushing; The glutaraldehyde of described linking agent to be volume fraction be 0.2-0.3%, crosslinking time is 24h;
5) being placed in vacuum drying oven drying by repeatedly rinsing to the spongy cross-linking products of neutral preparation, obtaining Composite collagen sponge, then after being cut into different size, oxirane disinfection, packaging, to obtain final product.
Embodiment five: the haemostatic effect experiment of Composite collagen sponge
This experiment is that hemostatic healing efficacy, hemostasis reliability and the body absorption situation to collagen sponge carries out preliminary study, and compares with solution Er Feisi.Animal used is 2.5 ~ 3.0kg Rabbit, and male and female are not limit.
Experimental technique is included in exempts from the hemorrhage surface of a wound that ear manufactures 2cm*1cm size, at rabbit liver, the splenectomy 1cm*1cm organization formation oozing of blood surface of a wound.After applying pressure hemostasis with sponge material, record bleeding stopping period, observes the bonding situation of dressing and the surface of a wound, and routine observation wound healing and body absorption situation.
Haemostatic effect is observed and contrast:
1) hemostatic healing efficacy is observed
The hemostasis of Biao Mian ear, liver and spleen.
In table, the bleeding stopping period of Composite collagen sponge of the present invention is shorter than and separates Er Feisi sheet, finds that the speed of Composite collagen sponge of the present invention absorption blood and the ability of bonding wound tissue are obviously better than separating Er Feisi sheet in experiment; Separate the phenomenon that 3 examples oozing of blood between dressing and tissue has then appearred in Er Feisi sheet.
2) hemostasis reliability
Manufacture 10 rabbit ear portion venous hemorrhage surface of a wound, apply pressure hemostasis by Composite collagen sponge and observe for latter 10 minutes, 24 hours, 48 hours, be showed no sponge and come off and bleeding again, separate Er Feisi sheet control group and occurred two routine sponge obscissions at 48 hours.10 livers, the spleen surface of a wound, collagen sponge applies pressure hemostasis and within latter 10 minutes, observes and all do not have hemorrhage and oozing of blood situation again, and sponge and the surface of a wound adhere to well; Within 24 hours, 48 hours, observations is the same.Sample sets all forms a complete coverture with sludged blood on the surface of a wound, sees that the surface of a wound has one deck red-purple tunica fibrosa after removing this coverture, nothing bleeding again.
3) wound healing and body absorption
Latter 1 week of rabbit ear portion surface of a wound hemostasis, collagen sponge coverture dries up gradually, comes off, and the surface of a wound is covered by new epithelize, and healing is good.Latter 2 weeks of rabbit liver, spleen hemostasis, observe the hemostasis coverture forming machine scar tissue of surface of a wound local, and have greater omentum tissue and its adhesion.After February, the surface of a wound is partially formed fiber sole mark, and collagen sponge is absorbed.
Conclusion: Composite collagen sponge bleeding stopping period of the present invention is short, the ability adhering to the surface of a wound is excellent, and haemostatic effect is obviously better than separating Er Feisi sheet; It spreads on surface wound, and the surface of a wound is dry, and not easily infect, wound healing is rapid; In body after internal organs hemostasis, local no abnormality seen irritant reaction, absorbed is good.
Embodiment six: the degradation rate experiment of Composite collagen sponge:
Take 5mg Composite collagen sponge respectively and conciliate Er Feisi sheet in 5m1 centrifuge tube, add 1m10.1MPBS (pH=7.4) and soak 1h, at 37 DEG C, add 0.05MCaC1
2with 200U collagenase, place 1d respectively, 3d, 5d, 7d, then centrifuge tube is put into ice bath to add 0.2m10.25MEDTA reaction is stopped, at 40 DEG C, centrifugal 15min under 5000r/min, get supernatant liquor and add 3m16MHCl be hydrolyzed 24h under room temperature, survey its absorbancy with ultraviolet spectrophotometer at 561nm place, obtain the concentration of oxyproline in its supernatant liquor according to hydroxyproline standard regression curve, ask its degradation rate with formula below:
Degradation rate (%)=Mq/M
t× 100%
Mq: the content of oxyproline in upper liquid; Mg/L
M
t: the quality of collagen in material; Mg
Each experiment is parallel does 3 times, and get its mean value, result is as follows:
| Degradation time | 1d | 3d | 5d | 7d |
| Separate Er Feisi sheet degradation rate (%) | 86.3 | 88.7 | 93.6 | 94.8 |
| The Composite collagen sponge degradation rate (%) of embodiment one | 31.6 | 38.2 | 46.8 | 77.2 |
| The Composite collagen sponge degradation rate (%) of embodiment two | 30.5 | 36.9 | 45.2 | 76.5 |
| The Composite collagen sponge degradation rate (%) of embodiment three | 32.4 | 37.5 | 45.3 | 75.8 |
| The Composite collagen sponge degradation rate (%) of embodiment four | 30.8 | 35.6 | 44.5 | 74.4 |
As can be known from the above table, under the corresponding time, the degradation rate of Composite collagen sponge lower than the degradation rate separating Er Feisi sheet, will overcome the feature that collagen is easily degraded to a certain extent.
Claims (3)
1. the preparation technology of a Composite collagen sponge, it is characterized in that: it is cross-linked by adding linking agent after a certain proportion of fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract lyophilize, through being washed to neutrality, secondary freeze drying and the soft spongy dressing of the porous made again; The mass ratio of described fish source collagen albumen, Bletilla glucomannan and Radix Lamiophlomidis Rotatae extract is 70-80:10-20:5-10, and its preparation technology comprises the following steps:
1) fish source collagen albumen being dissolved in volume fraction is in the acetic acid solution of 2%, makes the collagen solution that massfraction is 0.5%;
2) Bletilla glucomannan is immersed distilled water, after fully expanding, put in the water-bath of 50-60 DEG C and make it dissolve, be diluted with water to and make the Bletilla glucomannan solution that massfraction is 1-5%;
3) by above-mentioned obtained collagen solution and the mixing of Bletilla glucomannan solution, add Radix Lamiophlomidis Rotatae extract fine powder, stir;
4) by the mixing solutions moulding after mixing, obtain spongy material, spongy material is immersed linking agent and is cross-linked through lyophilize, cross-linking products is repeatedly extremely neutral through distilled water flushing; Glutaraldehyde or the concentration of described linking agent to be volume fraction be 0.2-0.3% are the EDC of 40-60mM, and crosslinking time is 12-24h;
5) being placed in vacuum drying oven drying by repeatedly rinsing to the spongy cross-linking products of neutral preparation, obtaining Composite collagen sponge, then after being cut into different size, oxirane disinfection, packaging, to obtain final product.
2. the preparation technology of Composite collagen sponge according to claim 1, is characterized in that the preparation technology of described fish source collagen albumen is: following all operations all carries out under lower than the condition of 20 DEG C; The NaOH solution of the fish skin raw material 0.1mol/L after thawing is soaked 24h and removes foreign protein, subsequently with deionized water fully wash to neutrality and with 10% n-butanol solvent soak degreasing 24h, with deionized water repetitive scrubbing, drain after add moment transformation extract fill with on high-pressure impregnation tank in, add the acetic acid solution of appropriate 0.5mol/L, inflating pressure 8 ~ 10kgf/cm
2, stir and soak 1-2 hour; The low pressure put into by material in high-pressure impregnation tank on the high-pressure impregnation tank of moment transformation extraction filling is extracted and is filled with, the acetic acid solution adding 0.5mol/L stirs and extracts 12h, repeat extraction 2 times, extract residue to continue to extract 24h with stirring containing the pepsic 0.5mol/L acetic acid of mass volume ratio 1.0%, merge No. 3 extracting solution centrifugations, obtain crude extract; In crude extract, adding NaCl to salt concn is 0.9mol/L, filter after leaving standstill the 24h that saltouts, use acetic acid and the distill water dialysis of 0.1mol/L after the acetic acid solution redissolution of collagen deposit 0.5mol/L successively, after last lyophilize, obtain fish source collagen protein sample;
The low pressure extraction filling comprising and be provided with agitator is filled with in described moment transformation extraction, and low pressure is extracted filling and is provided with the high-pressure impregnation tank connected by coupling cock, and described high-pressure impregnation tank top is provided with closed opening for feed, tensimeter and is connected the intake valve of air pump; Described low pressure is extracted the tank body of filling with and is provided with closed opening for feed, slag notch and baiting valve.
3. the preparation technology of Composite collagen sponge according to claim 1, is characterized in that the preparation technology of described Bletilla glucomannan is: by the bletilla striata, and pulverize, add 50-60 DEG C of water soaking 6-8h of 6-8 times amount, stir constantly, filter, filtrate is for subsequent use; The 50-60 DEG C of water soaking 2 times of dregs of a decoction 4-6 times amount again, soaks 4h at every turn, filters; Merge 3 filtrates, be condensed into paste, add 95% ethanol after cooling, make alcohol content reach 70%-75%, stir, namely separate out a large amount of cotton-shaped heavy liquid, leave standstill 1h, vacuum filtration is to dry, and vacuum-drying, porphyrize, to obtain final product.
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| CN104027835B (en) * | 2014-05-28 | 2015-08-12 | 中国科学院烟台海岸带研究所 | A kind of Aquatic product collagen protein of fish skin receives suction and preparation method thereof |
| CN106729984A (en) * | 2016-12-22 | 2017-05-31 | 青岛琛蓝海洋生物工程有限公司 | A kind of Isin glue collagen repairs sponge and preparation method thereof |
| CN108240923A (en) * | 2016-12-27 | 2018-07-03 | 中国科学院宁波城市环境观测研究站 | A kind of air sampling sponge and preparation method thereof |
| CN109847111B (en) * | 2018-12-21 | 2021-07-30 | 北京颐方生物科技有限公司 | Anti-adhesion material containing bletilla striata polysaccharide and preparation method thereof |
| CN110585474B (en) * | 2019-09-29 | 2022-01-04 | 青岛大学附属医院 | Preparation method of marine organism polysaccharide-based composite sponge, composite sponge and application |
| CN110665053B (en) * | 2019-10-31 | 2022-04-15 | 瑞研创展(清远)科技有限公司 | Medical composite collagen sponge dressing |
| CN111393521A (en) * | 2020-03-20 | 2020-07-10 | 张培华 | Extraction method of jellyfish collagen |
| CN114106148B (en) * | 2020-08-31 | 2023-12-01 | 中国海洋大学 | Medical-grade non-denatured collagen with fish skin source and preparation method thereof |
| CN113663120B (en) * | 2021-09-28 | 2022-10-28 | 桂林福康森医疗器械有限公司 | Hemostatic sponge cushion core and preparation method thereof |
| CN114149610A (en) * | 2021-12-17 | 2022-03-08 | 四川大学 | Method for preparing hemostatic sponge by using oxidized bletilla striata modified collagen fibers |
| CN116948241B (en) * | 2023-05-19 | 2024-03-29 | 南京普立蒙医疗科技有限公司 | Preparation method of frozen cross-linked protein sponge |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101648002A (en) * | 2008-08-13 | 2010-02-17 | 北京和润创新医药科技发展有限公司 | Medicinal composition for treating skin injury and ulcer and application thereof |
| CN102274493A (en) * | 2011-08-30 | 2011-12-14 | 董萍 | Bleeding-stopping, inflammation-diminishing and pain-relieving nano emulsion for use in minimally invasive beauty treatment therapy and preparation method thereof |
| CN103408787A (en) * | 2013-07-29 | 2013-11-27 | 武汉轻工大学 | Sponge material of collagen from freshwater fish and preparation method thereof |
-
2014
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101648002A (en) * | 2008-08-13 | 2010-02-17 | 北京和润创新医药科技发展有限公司 | Medicinal composition for treating skin injury and ulcer and application thereof |
| CN102274493A (en) * | 2011-08-30 | 2011-12-14 | 董萍 | Bleeding-stopping, inflammation-diminishing and pain-relieving nano emulsion for use in minimally invasive beauty treatment therapy and preparation method thereof |
| CN103408787A (en) * | 2013-07-29 | 2013-11-27 | 武汉轻工大学 | Sponge material of collagen from freshwater fish and preparation method thereof |
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