CN103435837B - Preparation method of recombinant human-like collagen biological sponge - Google Patents
Preparation method of recombinant human-like collagen biological sponge Download PDFInfo
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- CN103435837B CN103435837B CN201310403309.0A CN201310403309A CN103435837B CN 103435837 B CN103435837 B CN 103435837B CN 201310403309 A CN201310403309 A CN 201310403309A CN 103435837 B CN103435837 B CN 103435837B
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- Medicinal Preparation (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention discloses a preparation method of collagen biological sponge. The preparation method comprises the following steps: adding chitosan-containing acid liquor into collagen-containing acid liquor; uniformly mixing the liquor; injecting the liquor to a mould; freezing and moulding at low temperature; freezing and drying the frozen and moulded product; then, soaking the product in procyanidine-containing liquor to crosslink; and freezing and drying after washing to obtain the collagen biological sponge. The biological sponge disclosed by the invention has the advantages of controllability in moulding, no impurities, adaptability to microcellular structures, good physical and chemical properties, good biocompatibility and no animal collagen sponge virus hidden dangers, and is stable in quality and low in cost.
Description
Technical field
The invention belongs to biological medicine technology field, particularly, the present invention relates to a kind of preparation method of collagen protein bio-sponge and prepared bio-sponge thereof.
Background technology
At present conventional collagen protein is from the animal tissuess such as pigskin, ox-hide, ox bone, fish-skin, fish scale, to extract and obtain, molecular weight is uncertain, have a very wide distribution, and has animal virus hidden danger, can produce the shortcoming and defect such as rejection of allosome or xenogenesis again while being applied to human body.The inventor's early-stage Study has overcome this defect: utilize people's derived collagen albumen that gene recombination technology is produced to replace natural collagen.Recombination human source collagen protein is based on human III type collagen protein α 1 chain collagen domain Gly-X-Y(glycine-X-Y) tripeptides tumor-necrosis factor glycoproteins feature, design one section of people's derived collagen protein gene monomer of synthetic, by a series of vitro enzyme, cut connection, structure contains the expression vector of six series connection people derived collagen protein gene monomers in the same way, proceed to pichia pastoris bacterium, by high density fermentation, obtain with separated, purifying.
The collagen bio-sponge that possesses mesh structural porous structure is substrate material important in organizational project, can analog cell epimatrix, be beneficial to cell adhesion and infiltration, guide cytodifferentiation and propagation to form functional destination organization, and institute's carrying medicament is had to slow releasing function, thereby there is significant application value aspect analog cell tumor growth environment, the filling of setting up in-vitro cell culture model or promotion wound, reparation, regeneration.
Utilize pycnogenols as linking agent, contribute to improve the mechanical property of collagen protein and chitosan copolymerization sponge (or support, film), being about to collagen protein and chitosan is dissolved in acidic solution altogether, add pycnogenols to carry out crosslinking reaction, then pack die for molding into, but generally can only form film shape (referring to Lin Xuming, etc. the Study on biocompatibility of taking proanthocyanidins crosslinked timbering material. Recent Advances in Ophthalmology .27 (4): 262; Dang Meizhu. preparation and the applied research thereof of natural polyphenol modification fish scale collagen-chitosan film matrix material. Hua Zhong Agriculture University's academic dissertation collection, etc.).
Prior art is after bio-sponge moulding, or directly use, or in room temperature or high temperature drying, if wherein need to remove, utilizes the acid of introducing while preparing bio-sponge, adds the alkaline matters such as sodium hydroxide, ammonia to neutralize.Yet the former can only prepare use temporarily, be unfavorable for the Industry Promotion application of bio-sponge; And studying discovery through the present invention, the latter needs the strict amount of controlling the pycnogenols adding, otherwise is unfavorable for the mechanical property of bio-sponge.In addition, by adding alkaline matter to neutralize, although pH value can reach physiological requirement, can make to remain the salt such as ammonium salt, sodium salt in bio-sponge, affect its use range, contact in vivo or directly in the use of wound and be greatly limited.
The inventor, through studying for a long period of time, has chanced on a kind of preparation flow, and first, by collagen protein and chitosan lyophilize moulding, the pycnogenols solution re-using containing ethanol is cross-linked, lyophilize again after washing.Whole process can be used current mature equipment, preparation process is insensitive to procyanidin concentration, cost less investment, and can improve the mechanical property (the not crumple if support fluffy bulk) of bio-sponge, especially formed product is good, completely according to mould molding, form size, size, the controlled sponge of shape, especially can form non-film like, in this flow process, can remove acidic substance in addition, do not leave the salt such as ammonium salt, sodium salt residual, and be easy to preserve, be convenient to Industry Promotion widely.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation method of new recombination human source collagen protein bio-sponge.In addition, the present invention also provides the bio-sponge of being prepared by the method.
Particularly, in first aspect, the invention provides a kind of preparation method of collagen protein bio-sponge, it comprises:
(1) acidic solution of chitosan-containing is joined in the acidic solution containing collagen protein, after mixing, inject mould, cryogenic freezing moulding;
(2) the freeze forming product of step (1) is carried out to lyophilize, obtain elementary bio-sponge;
(3) elementary bio-sponge step (2) being obtained is soaked into containing crosslinked in the solution of pycnogenols, then washes with water;
(4) washed product step (3) being obtained is carried out lyophilize, obtains collagen protein bio-sponge; With,
(5) optionally collagen protein bio-sponge is cut out.
In this article, collagen protein is preferably recombination human source collagen protein.For example, recombination human source collagen protein of the present invention can be the recombination human source collagen protein that the disclosed method of Chinese patent 200610098297.5 obtains, more preferably, the high density fermentation providing by patent publication No. CN102443057A and purification process, by the constructed pichia pastoris genetic engineering bacterium of different repeat number series aiding connection gene recombination plasmids, (culture presevation is in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: the recombination human source collagen protein of CGMCC NO. 5021) producing in utilization.People's derived collagen protein ratio plays animal (especially nonmammalian) collagen protein, more reliable to the safety in utilization of human body, although physical strength can be weaker.
In this article, low temperature is lower than-10 ℃.Conventionally utilize liquid nitrogen, dry ice can form stable low temperature, in certainly commercially available refrigerator, have can stable maintenance-80 ℃ refrigerator, also can adopt.So preferably, in the preparation method of a first aspect of the present invention, the low temperature in step (1) is-20 ~-195 ℃, preferably-20 ~-80 ℃, as-80 ℃.
Preferably in the preparation method of a first aspect of the present invention, collagen protein after mixing in step (1) and the mass ratio of chitosan are 0.05 ~ 10:1,0.1 ~ 9:1 preferably, being more preferably is the high corresponding mass ratio of physical strength in the embodiment of the present invention.Some preferred embodiments, block in the situation that, have been equivalent to the physical strength of the film like that density prepared by prior art is higher.
Preferably in the preparation method of a first aspect of the present invention, in the acidic solution of the chitosan-containing in step (1), the content of chitosan is 0.05 ~ 7%(w/w), be preferably 0.1 ~ 5%(w/w), being more preferably is the high corresponding content of physical strength in the embodiment of the present invention.Chitosan can obtain easily by commercially available channel.
Preferably in the preparation method of a first aspect of the present invention, the content containing collagen protein in the acidic solution of collagen protein in step (1) is 0.05 ~ 7%(w/w), be preferably 0.1 ~ 5%(w/w), being more preferably is the high corresponding content of physical strength in the embodiment of the present invention.
The inventor studies discovery, if do not add ethanol, even through freezing step, and also can the have an appointment decline of an order of magnitude of physical strength.So, preferably, in the preparation method of a first aspect of the present invention, the solution in step (3) is containing dehydrated alcohol, and wherein the content of dehydrated alcohol is 5 ~ 60%(V/V), be preferably 10 ~ 50%(V/V), being more preferably is the high corresponding content of physical strength in the embodiment of the present invention.
In this article, acidic solution is acetic acid solution preferably, as 0.3 ~ 7%(w/w) acetic acid solution, preferably 0.5 ~ 5%(w/w) acetic acid solution, being more preferably is the high corresponding acetic acid solution of physical strength in the embodiment of the present invention.Preferably, in the preparation method of a first aspect of the present invention, the pH value of the solution in step (3) is 5 ~ 9.5, is preferably 6 ~ 9, and being more preferably is the high corresponding pH of physical strength in the embodiment of the present invention.
In this article, being soaked in the solution containing pycnogenols is to be soaked into excessive containing in the solution of pycnogenols, and before and after soaking, in solution, the content of pycnogenols is substantially constant, as front and back concentration only declines and is no more than 10%, or only declines and is no more than 5%.This container by larger splendid attire solution a large amount of solution that injects can be realized.Inventor's discovery, procyanidin concentration has certain influence for the physical strength of bio-sponge.Preferably in the preparation method of a first aspect of the present invention, the content of the pycnogenols of the solution in step (3) is 0.01 ~ 5%(w/w), be preferably 0.05 ~ 4%(w/w), 0.08 ~ 3%(w/w more preferably), most preferably be 0.1 ~ 2%(w/w), corresponding content as high in physical strength in the embodiment of the present invention.
In this article, washing is in order to remove not reactant, as salt and soda acid.In practice, the pH after washing be washing can operation index, bio-sponge is washed neutral to pH or approaches neutrally, can represent completing of washing.Preferably, in the preparation method of a first aspect of the present invention, the washing in step (3) is to wash to pH6.5 ~ 7.5, preferably 6.8 ~ 7.2.
In this article, bio-sponge of the present invention can formalize according to the shape of mould.By the size and shape of mould, can make according to the actual requirements arbitrary shape and size, as rectangular parallelepiped, cubes, right cylinder, truncated cone-shaped, etc.And prepared by prior art, be film like (or claim " film ") substantially, thickness is generally no more than 0.1cm, even can printing opacity.Preferably in the preparation method of a first aspect of the present invention, collagen protein bio-sponge is not film like, preferably lighttight, be more preferably block, as (being less than long and/or wide) of preferred bulk is highly greater than 0.1cm, be preferably not less than 0.2cm, be more not less than 0.5cm, be more preferably and be not less than 1cm, as be not less than 2cm, 3cm or 5cm.
In second aspect, the invention provides collagen protein bio-sponge prepared by the preparation method of a first aspect of the present invention.Preferably collagen protein bio-sponge is not film like, preferably lighttight, is more preferably block, as (being less than long and/or wide) of preferred bulk is highly greater than 0.1cm, be preferably not less than 0.2cm, be more not less than 0.5cm, be more preferably and be not less than 1cm, as be not less than 2cm, 3cm or 5cm.The collagen protein bio-sponge that provides in embodiments of the present invention a large amount of different methods to prepare, is more preferably that wherein physical strength is high.
In the third aspect, the application of the collagen protein bio-sponge that the invention provides a second aspect of the present invention in the pharmaceutical carrier of the non-instant use of preparation.Wherein, preferred agents carrier is weighting material and/or restoration or the tissue engineering bracket of drug release carrier, tissue and/or wound.Because bio-sponge plasticity of the present invention is good, the leeway that has facilitated direct use or cut out, so the adaptability of using is good; In addition free from foreign meter, very big degree is avoided in the side effect causing or untoward reaction.These advantages are that prior art cannot be reached.
Beneficial effect of the present invention is, recombination human source collagen protein bio-sponge of the present invention, plasticity is good, can make arbitrary size and shape (being not limited to film like), and free from foreign meter, there is in addition suitable microvoid structure, physicochemical property is good, biocompatibility is good, can be for surgical operation, build cells in vitro dimensional culture model, the filling of preparation drug release apparatus, tissue and wound and reparation apparatus, tissue engineering bracket; In addition, its preparation method is quality controllable and stable, with low cost.
For the ease of understanding, below will to the present invention, be described in detail by specific embodiment and accompanying drawing.It needs to be noted, specific examples is only in order to illustrate, does not form limitation of the scope of the invention.Obviously those of ordinary skill in the art can make various corrections and change to the present invention within the scope of the invention according to explanation herein, and these corrections and change are also included in scope of the present invention.
In addition, the present invention has quoted open source literature, and these documents are also in order more clearly to describe the present invention, and their full text content is all included the present invention in and carried out reference, just look like they full text in specification sheets of the present invention repeated description excessively the same.
Accompanying drawing explanation
Fig. 1 and Fig. 2 have shown bio-sponge of the present invention, and its shape can determine according to mould completely, can be prepared into very thick (at least can not printing opacity), are not limited to film like.
Embodiment
Below in conjunction with embodiment, the invention will be further described, wherein reagent used, raw material are all currently available productss, if collagen protein is according to the prepared recombination human source collagen protein of the early stage Chinese patent ZL 200610098297.5 of the inventor, chitosan can be purchased from life work biotechnology (Shanghai) limited-liability company, and pycnogenols can be purchased from Jianfeng Natural Product R&D Development Co., Ltd., Tianjin.
The preparation 1 of embodiment 1 recombination human source collagen protein bio-sponge
Recombination human source collagen protein is dissolved in to 3%(w/w) in acetic acid aqueous solution, be mixed with 5%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 3%(w/w) in acetic acid aqueous solution, be mixed with 5%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 10%(v/v) in the phosphate buffered saline buffer (pH6) of dehydrated alcohol, be mixed with 0.5%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan is while being 0.1:1, continue to stir 240 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, be placed in again vacuum freeze drier (the FDU-1200 model of Tokyo physics and chemistry apparatus company, with reference to its manufacturer's declaration condition, carry out lyophilize) middle lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reach 6.8 or more than, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 2 of embodiment 2 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 3%(w/w) in acetic acid aqueous solution, be mixed with 4%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 3%(w/w) in acetic acid aqueous solution, be mixed with 4%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 20%(v/v) in the phosphate buffered saline buffer (pH6) of dehydrated alcohol, be mixed with 2%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 0.25:1, continues to stir 180 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reach 6.8 or more than, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 3 of embodiment 3 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 2%(w/w) in acetic acid aqueous solution, be mixed with 2%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 2%(w/w) in acetic acid aqueous solution, be mixed with 2%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 20%(v/v) in the phosphate buffered saline buffer (pH6.8) of dehydrated alcohol, be mixed with 1%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 0.5:1, continues to stir 120 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reaches 7.0, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 4 of embodiment 4 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 4%(w/w) in acetic acid aqueous solution, be mixed with 1.5%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 4%(w/w) in acetic acid aqueous solution, be mixed with 1.5%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 30%(v/v) in the phosphate buffered saline buffer (pH7) of dehydrated alcohol, be mixed with 1.5%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 1:1, continues to stir 120 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 5 of embodiment 5 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 1%(w/w) in acetic acid aqueous solution, be mixed with 1%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 1%(w/w) in acetic acid aqueous solution, be mixed with 1%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 40%(v/v) in the phosphate buffered saline buffer (pH7.4) of dehydrated alcohol, be mixed with 0.5%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 1:1, continues to stir 60 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reaches 7.2 or following, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 6 of embodiment 6 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 1%(w/w) in acetic acid aqueous solution, be mixed with 1%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 1%(w/w) in acetic acid aqueous solution, be mixed with 1%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 40%(v/v) in the phosphate buffered saline buffer (pH7.4) of dehydrated alcohol, be mixed with 1%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 2:1, continues to stir 60 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reaches 7.2 or following, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 7 of embodiment 7 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 0.5%(w/w) in acetic acid aqueous solution, be mixed with 0.5%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 0.5%(w/w) in acetic acid aqueous solution, be mixed with 0.5%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 50%(v/v) in the phosphate buffered saline buffer (pH8) of dehydrated alcohol, be mixed with 0.2%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 4:1, continues to stir 30 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reaches 7.2 or following, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
The preparation 8 of embodiment 8 recombination human source collagen protein bio-sponges
Recombination human source collagen protein is dissolved in to 0.5%(w/w) in acetic acid aqueous solution, be mixed with 0.1%(w/w) recombination human source collagen solution; Chitosan is dissolved in to 0.5%(w/w) in acetic acid aqueous solution, be mixed with 0.1%(w/w) chitosan solution; In addition, pycnogenols is dissolved in containing 10%(v/v) in the phosphate buffered saline buffer (pH9) of dehydrated alcohol, be mixed with 0.1%(w/w) pycnogenols solution.
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 9:1, continues to stir 30 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge.Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in room temperature (25 ℃), then with deionized water, soak, change during this time water repeatedly (as, 3 times), until the pH of the water of wash-out reaches 7.2 or following, be again placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
Embodiment 9 comparative examples
The process preparation contrast bio-sponge that repeats above embodiment, difference is without lyophilize, but all at room temperature dries.The contrast of drying under room temperature all can crumple in drying process, is merely able to form film like, cannot maintain fluffy lumphy structure.
Bio-sponge prepared by above-described embodiment is cut into small pieces, and (detect thickness of contrast is got the thickness that the thickness that is equivalent to the embodiment of the present invention obtains with crumple after aforesaid method preparation, the method preparation that about 1cm thickness is optimized through non-the present invention, meeting crumple is into about 0.08cm), speed with 100mm/min after pressurized is fixing is at the uniform velocity stretched to Materials Fracture, the tensile strength of measuring above-described embodiment and contrast bio-sponge, result is as shown in table 1.Result shows, adopt the immersion of pycnogenols of the present invention crosslinked, can improve the physical strength of bio-sponge, especially for collagen protein and the large situation of chitosan ratio, increase rate is larger, and the physical strength of the lumphy structure that it is more fluffy can be equivalent to film like body that density prepared by prior art is higher.
The tensile strength of table 1 bio-sponge (MPa)
| ? | Bio-sponge | Elementary bio-sponge | Contrast bio-sponge | Contrast elementary bio-sponge |
| Embodiment 1 | 58.6 | 28.4 | 37.6 | 27.8 |
| Embodiment 2 | 61.7 | 27.2 | 42.2 | 28.1 |
| Embodiment 3 | 59.3 | 26.3 | 40.7 | 26.4 |
| Embodiment 4 | 56.9 | 26.1 | 37.1 | 24.7 |
| Embodiment 5 | 57.2 | 14.3 | 36.8 | 15.2 |
| Embodiment 6 | 56.0 | 14.1 | 35.7 | 12.9 |
| Embodiment 7 | 58.2 | 12.9 | 37.3 | 11.5 |
| Embodiment 8 | 55.3 | 13.7 | 34.5 | 13.6 |
Embodiment 10 toxicity tests
Vitro skin stimulates test: 4 health (skin without redness, oedema, breakage) rabbit back backbone both sides hair is removed to about 10cm2 area, the bio-sponge of getting embodiment 1 preparation sticks on left field, right side area does not stick in contrast, observes and sticks the skin after 1,12,24,48,72 hour.In the above period, the region that sticks of all rabbits all produces without phenomenons such as redness, oedema, breakage, pigmentation, skin roughen or attenuation, and wherein have the region that do not stick of a rabbit to produce disrepair phenomenon, estimate that this is because Dermal exposure is subject to due to physical abuse outside.Result shows, the external use safety non-toxic of bio-sponge of the present invention, and especially permeability is good, even if reach three days, sticks, and also skin is not caused to untoward reaction.
Cytotoxicity test: get fell venous endothelial cell (can purchased from Chinese Academy of Sciences's Shanghai cell bank), be placed in containing the DMEM substratum of 10% foetal calf serum and cultivate, the fritter of 5mm * 5mm * 25mm will be cut into according to the method described in standard GB/T/T16886.12-2005 after the bio-sponge Co60 sterilizing of embodiment 1 preparation, in the lixiviate ratio of 1.25cm2/mL, be soaked in containing in the DMEM substratum of 10% foetal calf serum, 37 ℃ of lixiviate 24h, get the substratum after lixiviate, according to the method described in GB/T16886.5-2003, carry out cell cultures, the DMEM substratum of blank 10% foetal calf serum containing not soaking in contrast.With control group contrast, test group does not find to have difference in the attached cell speed of growth, quantity and form, shows that bio-sponge of the present invention is nontoxic to cells in vivo.
Claims (1)
1. a preparation method for collagen protein bio-sponge, it is:
Recombination human source collagen protein is dissolved in 3% (w/w) acetic acid aqueous solution, is mixed with 4% (w/w) recombination human source collagen solution; Chitosan is dissolved in 3% (w/w) acetic acid aqueous solution, is mixed with 4% (w/w) chitosan solution; In addition, pycnogenols is dissolved in the phosphate buffered saline buffer of the pH=6 that contains 20% (v/v) dehydrated alcohol, is mixed with 2% (w/w) pycnogenols solution;
Under condition of ice bath, chitosan solution is splashed into recombination human source collagen solution, stir during this time, until the mass ratio of recombination human source collagen protein and chitosan while being 0.25:1, continues to stir 180 minutes, with the vacuum defoamation of vacuum defoamation machine, then mixing solutions is injected to mould, freeze forming at-80 ℃ of temperature, then be placed in vacuum freeze drier lyophilize, form elementary bio-sponge;
Then, elementary bio-sponge is soaked in pycnogenols solution and is cross-linked 48 hours in 25 ℃, then with deionized water, soak, change during this time water repeatedly, until the pH of the water of wash-out reach 6.8 or more than, again be placed in vacuum freeze drier lyophilize, obtain recombination human source collagen protein bio-sponge.
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