CN1179758C - Preparation method of collagen/chitosan porous scaffold for tissue engineering - Google Patents
Preparation method of collagen/chitosan porous scaffold for tissue engineering Download PDFInfo
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- CN1179758C CN1179758C CNB021124981A CN02112498A CN1179758C CN 1179758 C CN1179758 C CN 1179758C CN B021124981 A CNB021124981 A CN B021124981A CN 02112498 A CN02112498 A CN 02112498A CN 1179758 C CN1179758 C CN 1179758C
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Abstract
The present invention discloses a porous scaffold which is prepared by adopting natural biomaterial collagen and chitosan as raw materials by freezing-freeze drying with a vacuum dry heating method further and by crosslinking aldehyde compounds or carbodiimide compounds, and has the advantages of controllable microstructure, suitable degradation rate and favorable biocompatibility. The collagen/chitosan scaffold of the present invention has the advantages of good biocompatibility, controllable degradation rate, wide material sources, low cost, simple and feasible preparation technology and good repeatability. The constructed scaffold can be widely applied to the field of tissue engineering, and has a favorable prospect of clinical application.
Description
Technical field
The present invention relates to the preparation method of used in tissue engineering collagen/chitosan porous rack, relate to the preparation method of the histocompatibility three-dimensional porous rack that is used for transmitting tissue and neomorph specifically.
Background technology
Organizational project is one and relates to multidisciplinary, multi-field crossing research problems such as medical science, chemistry, biology, materialogy, and the structure of three-dimensional rack is one of key wherein.Only construct have specific microstructure, good mechanical performance, suitable degradation property and the support of excellent biological compatibility, could promote the adhesion and the growth of cell effectively, and then the regeneration of transmitting tissue and organ.
Collagen is the main component in the mammal connective tissue, constitutes the protein of human body about 30%, and the dry weight in skin reaches 72%.Collagen has 19 types, and modal is I type, II type and III type.Wherein the I type is the abundantest, and function admirable, is widely used in biomaterial.Though collagen-based materials has unrivaled biocompatibility, but the mechanical strength of the support that makes up with pure collagen is lower, degradation rate is too fast, can not satisfy the requirement of tissue engineering bracket, therefore it is also crosslinked with acquisition and the similar supporting structure of extracellular matrix by suitable method with the performance of improving collagen scaffold to be necessary to add other component, and has the degradation rate that is complementary with tissue regeneration.
Chitosan is a kind of polysaccharose substance that has polyamino, the main component glycosaminoglycan of its structure and some character and extracellular matrix is similar, have excellent biological compatibility and suitable degradation property, nonirritant, non-immunogenicity, no heat source response, and have the function that promotes wound healing, and wide material sources, with low cost.Chitosan has been widely used in suture, Wound dressing and the organizational project three-dimensional rack at present.
Summary of the invention
The preparation method that the purpose of this invention is to provide a kind of used in tissue engineering collagen/chitosan porous rack.
The present invention is to be material with collagen and chitosan, prepares the used in tissue engineering porous support by freezing-lyophilization, and adopts the method for physics and chemistry to carry out cross-linking modified to porous support.This preparation method may further comprise the steps:
1) respectively under acid condition compound concentration (by weight) be 0.1%～5%, be preferably 0.3%～3% collagen solution and chitosan solution, chitosan solution is splashed in the collagen solution, stir the collagen/chitosan mixed solution, the content of chitosan solution (by weight) is 1～80%, is preferably 5～30%;
2) the collagen/chitosan blended liquid is injected mould, adopts freezing-lyophilization, at-10 ℃～-100 ℃, be preferably under-10 ℃～-50 ℃ temperature after freezing 0.05～5 hour, lyophilizing in freeze dryer must the collagen/chitosan three-dimensional porous rack;
3) with step 2) gained collagen/chitosan three-dimensional porous rack is crosslinked with vacuum xeothermic physical method, crosslinking temperature is 80～130 ℃, time is 1～48 hour, soaked 1～3 hour at acetic acid solution then, put into aldehyde compound or the chemical crosslinking of carbodiimides compounds 1～48 hour again;
4) clean freezing once more, lyophilizing.
Among the present invention, the collagen-based materials of preparation collagen solution is beef tendon collagen, pigskin collagen, Mus tail collagen.Preparation collagen solution and the used acid of chitosan solution are a kind of or its mixture in acetic acid, formic acid, the hydrochloric acid.The pH value of said collagen/chitosan mixed liquor is 2～5.
Among the present invention, aldehyde compound is formaldehyde or glutaraldehyde.The concentration of aldehyde compound is 0.01%～2.5%, and preferred concentration is 0.05～0.25%.
Among the present invention, the carbodiimides compounds is meant 1-ethyl-3-3 dimethyl amine propyl group-carbodiimides (EDAC).The Carbodiimides compound concentrations is the aqueous solution of 0.001～1M, and preferred concentration is 0.01～0.3M.
Molecular weight to said chitosan material among the present invention does not have special requirement, is 1～2,000,000 chitosan but preferably use molecular weight ranges.
The material that the inventive method adopted belongs to natural biologic material, has excellent biological compatibility and degradability, and material source is extensive, and is with low cost.Adopt freezing-lyophilization can pass through the micro structure of the factor may command porous supports such as blend ratio of adjusting cryogenic temperature, blended liquid concentration or pH value, collagen and chitosan, prepare three-dimensional porous rack with suitable aperture and porosity.The employing chitosan is a bridging agent, and is crosslinked to regulate the degradation rate of porous support through the method for physics or chemistry.Be prepared into reconstruction or the regeneration that porous support can be widely used in multiple tissues such as skin, cartilage, bone, blood vessel, nerve, tendon, cardiac valve or organ by the present invention, with the external structure or the internal regeneration of above-mentioned tissue of effective promotion or organ.
Description of drawings
Fig. 1 a chitosan concentration is 10% o'clock, the SEM photo of support;
Fig. 1 b chitosan concentration is 50% o'clock, the SEM photo of support;
Fig. 2 a cryogenic temperature is the SEM photo of the support for preparing under-50 ℃ of conditions;
Fig. 2 b cryogenic temperature is the SEM photo of the support for preparing under-20 ℃ of conditions;
The chitosan concentration of the glutaraldehyde cross-linking of Fig. 3 variable concentrations is the degradation property of 10% beef tendon collagen/chitosan support;
The chitosan concentration of Fig. 4 0.25% glutaraldehyde cross-linking is 10% the laser confocal microscope photo of beef tendon collagen/chitosan support plantation 3T3 cell after 7 days;
The chitosan concentration of Fig. 5 a 0.25% glutaraldehyde cross-linking is 10% 3 days tissue slice figure of the beef tendon collagen/chitosan support implantation rabbit ear back of the body;
Fig. 5 b is that the chitosan concentration of 0.25% glutaraldehyde cross-linking is 10% 7 days tissue slice figure of the beef tendon collagen/chitosan support implantation rabbit ear back of the body;
Fig. 5 c is that the chitosan concentration of 0.25% glutaraldehyde cross-linking is 10% 14 days tissue slice figure of the beef tendon collagen/chitosan support implantation rabbit ear back of the body;
Fig. 5 d is that the chitosan concentration of 0.25% glutaraldehyde cross-linking is 10% 28 days tissue slice figure of the beef tendon collagen/chitosan support implantation rabbit ear back of the body;
Fig. 6 is that the chitosan concentration of 0.25% glutaraldehyde cross-linking is the pigskin collagen/laser confocal microscope photo of chitosan stent plantation 3T3 cell after 7 days of 10%;
The specific embodiment
Embodiment 1: chitosan content is to the influence of the micro structure of beef tendon collagen/chitosan porous rack
The method that adopts enzymolysis to combine with sour extracting is extracted from beef tendon and is obtained beef tendon collagen.With acetic acid solution respectively compound concentration be that 0.5% collagen solution and concentration are 0.5% chitosan solution, then chitosan solution and the even blend of collagen solution are obtained the collagen/chitosan mixed solution that chitosan content is respectively (by weight) 10%, 20%, 30%, 50%.In-20 ℃ freezing 1 hour, can prepare porous support after the lyophilizing with different apertures and pattern, see Fig. 1 a, Fig. 1 b.Fig. 1 a, Fig. 1 b are respectively that chitosan content is scanning electron microscope (SEM) photo of 10%, 50% collagen/chitosan support.
Embodiment 2: cryogenic temperature is to the influence of the micro structure of beef tendon collagen/chitosan porous rack
The method that adopts enzymolysis to combine with sour extracting is extracted from beef tendon and is obtained beef tendon collagen.With the acetic acid solution of 0.5M respectively compound concentration be 0.5% collagen solution and 0.5% chitosan solution, then chitosan solution and the even blend of collagen solution being obtained chitosan content is the collagen/chitosan mixed solution of (by weight) 10%.Then respectively at-50 ℃ ,-20 ℃ freezing 1 hour, can prepare the different porous support of pore size after the lyophilizing, see Fig. 2 a, Fig. 2 b.Fig. 2 a, Fig. 2 b are respectively at-50 ℃, the SEM photo of the porous support of cryopreparation under-20 ℃ of temperature.
Embodiment 3: beef tendon collagen/chitosan porous rack crosslinked
The method that adopts enzymolysis to combine with sour extracting is extracted from beef tendon and is obtained beef tendon collagen.With the acetic acid solution of 0.5M respectively compound concentration be that 0.5% collagen solution and concentration are 0.5% chitosan solution, then chitosan solution and the even blend of collagen solution being obtained chitosan content is the collagen/chitosan mixed solution of (by weight) 10%.Adopt freezing-lyophilization, in-20 ℃ freezing 1 hour, lyophilizing in 24 hours in the freeze dryer then.Xeothermic crosslinked 24 hours of freeze dried collagen/chitosan support vacuum in 105 ℃ vacuum drying oven obtains xeothermic crosslinked beef tendon collagen/chitosan porous rack.Through the beef tendon collagen/chitosan porous rack of the xeothermic crosslinked acquisition of vacuum soaks 1 hour in the acetic acid solution of 100ml 0.5M after, in 4 ℃ crosslinked 24 hours down with 0.05%～0.25% glutaraldehyde solution, repeatedly after the rinsing, freezing once more-lyophilizing obtains the different collagen/chitosan porous rack of the degree of cross linking with tri-distilled water.The degradation resistant performance of crosslinked back beef tendon collagen/chitosan porous rack is significantly improved, and sees Fig. 3.
Embodiment 4: beef tendon collagen/chitosan porous rack in-vitro evaluation
The method that adopts enzymolysis to combine with sour extracting is extracted from beef tendon and is obtained beef tendon collagen.With the acetic acid solution of 0.5M respectively compound concentration be that 0.5% collagen solution and concentration are 0.5% chitosan solution, then chitosan solution and the even blend of collagen solution being obtained chitosan content is the collagen/chitosan mixed solution of (by weight) 10%.Adopt freezing-lyophilization, in-20 ℃ freezing 1 hour, lyophilizing in 24 hours in the freeze dryer then.Xeothermic crosslinked 24 hours of freeze dried collagen/chitosan support vacuum in 105 ℃ vacuum drying oven.The xeothermic crosslinked porous support of vacuum soaked in the acetic acid solution of 100ml 0.5M after 1 hour, in 4 ℃ crosslinked 24 hours down with 0.25% glutaraldehyde solution, repeatedly after the rinsing, freezing once more-the crosslinked collagen/chitosan porous rack of lyophilizing acquisition with tri-distilled water.3T3 cell (5,000,000/ml), 37 ℃ of following 5%CO of plantation 1ml in this porous support
2Cultivated in the incubator 7 days, the next day change liquid.Fluorescein diacetate (FDA) dyeing back laser confocal microscope (CLSM) is observed under rack surface visible a large amount of 3T3 cells in the 200 μ m, and cell growth state is good, sees Fig. 4.
Embodiment 5: beef tendon collagen/chitosan porous rack interior evaluating
The method that adopts enzymolysis to combine with sour extracting is extracted from beef tendon and is obtained beef tendon collagen.With the acetic acid solution of 0.5M respectively compound concentration be that 0.5% collagen solution and concentration are 0.5% chitosan solution, then chitosan solution and the even blend of collagen solution being obtained chitosan content is the collagen/chitosan mixed solution of (by weight) 10%.Adopt freezing-lyophilization, in-20 ℃ freezing 1 hour, lyophilizing in 24 hours in the freeze dryer then.Xeothermic crosslinked 24 hours of freeze dried collagen/chitosan support vacuum in 105 ℃ vacuum drying oven.The xeothermic crosslinked porous support of vacuum soaked in the acetic acid solution of 100ml 0.5M after 1 hour, in 4 ℃ crosslinked 24 hours down with 0.25% glutaraldehyde solution, repeatedly after the rinsing, freezing once more-the crosslinked collagen/chitosan porous rack of lyophilizing acquisition with tri-distilled water.Be imbedded at the full thick skin wound surface of rabbit of artificial excision after the support sterilization,, 7 days, 14 days, make tissue slice after 28 days and observe respectively at after the heeling-in 3 days.Heeling-in has a small amount of inflammatory cell to exist after 3 days.Visible fibroblast is grown into after 7 days, and supporting structure still keeps, and does not see tangible inflammatory cell in the support, after 14 days, had a large amount of fibroblastic growths to enter in the support, and support has had vascularization to a certain degree.Heeling-in has been merged intact in surrounding tissue after 28 days, be the dermal tissue sample, sees Fig. 5 a, Fig. 5 b, Fig. 5 c, Fig. 5 d.Fig. 5 a, Fig. 5 b, Fig. 5 c, Fig. 5 d are respectively the collagen/chitosan support and implanted 3 days, and 7 days, 14 days, 28 days tissue slice figure.
Embodiment 6: pigskin collagen/chitosan porous rack makes up and in-vitro evaluation
The method that adopts enzymolysis to combine with sour extracting is extracted from fresh porcine skin and is obtained pigskin collagen.With the acetic acid solution of 0.5M respectively compound concentration be that 0.5% collagen solution and concentration are 0.5% chitosan solution, then chitosan solution and the even blend of collagen solution being obtained chitosan content is the collagen/chitosan mixed solution of (by weight) 10%.Adopt freezing-lyophilization, in-20 ℃ freezing 1 hour, lyophilizing in 24 hours in the freeze dryer then.Xeothermic crosslinked 24 hours of freeze dried collagen/chitosan support vacuum in 105 ℃ vacuum drying oven, the xeothermic crosslinked porous support of vacuum soaks after 1 hour in the acetic acid solution of 100ml 0.5M, in 4 ℃ crosslinked 24 hours down with 0.25% glutaraldehyde solution, repeatedly after the rinsing, freezing once more-lyophilizing obtains crosslinked collagen/chitosan porous rack with tri-distilled water.3T3 cell (5,000,000/ml), 37 ℃ of following 5%CO of plantation 1ml in this porous support
2Cultivated in the incubator 7 days, the next day change liquid.FDA dyeing back CLSM observes on the visible hole wall in support and is pasted with a large amount of 3T3 cells, and cell is the rounded grain shape, and the as seen existing intercellular substance of iuntercellular exists, and sees Fig. 6.
Claims (12)
1. the preparation method of used in tissue engineering collagen/chitosan porous rack is characterized in that it may further comprise the steps:
1) respectively under acid condition the preparation weight concentration be 0.1%～5% collagen solution and chitosan solution, chitosan solution is splashed in the collagen solution, stir the collagen/chitosan mixed solution, the weight content of chitosan solution is 1～80%;
2) the collagen/chitosan blended liquid is injected mould, adopt freezing-lyophilization, after under-10 ℃～-100 ℃ temperature freezing 0.05～5 hour, lyophilizing in freeze dryer gets the collagen/chitosan three-dimensional porous rack;
3) with step 2) gained collagen/chitosan three-dimensional porous rack is crosslinked with vacuum xeothermic physical method, crosslinking temperature is 80～130 ℃, time is 1～48 hour, soaked 1～3 hour at acetic acid solution then, put into aldehyde compound or the chemical crosslinking of carbodiimides compounds 1～48 hour again;
4) clean freezing once more, lyophilizing.
2. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, the collagen-based materials that it is characterized in that preparing collagen solution is beef tendon collagen, pigskin collagen, Mus tail collagen.
3. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, it is characterized in that preparing collagen solution and the used acid of chitosan solution and be a kind of or its mixture in acetic acid, formic acid, the hydrochloric acid.
4. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, it is characterized in that said collagen/chitosan mixed liquor, the weight content of its chitosan solution is 5～30%.
5. press the preparation method of claims 1 described used in tissue engineering collagen/chitosan porous rack, the pH value that it is characterized in that said collagen/chitosan mixed liquor is 2～5.
6. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, it is characterized in that said aldehyde compound is formaldehyde or glutaraldehyde.
7. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, it is characterized in that the carbodiimides compounds is meant 1-ethyl-3-3 dimethyl amine propyl group-carbodiimides.
8. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, it is characterized in that step 2) cryogenic temperature be-10 ℃～-50 ℃.
9. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, the concentration that it is characterized in that said aldehyde compound is 0.01%～2.5%.
10. by the preparation method of the described used in tissue engineering collagen/chitosan porous rack of claim 1, the concentration that it is characterized in that said aldehyde compound is 0.05%～0.25%.
11. press the preparation method of claims 1 described used in tissue engineering collagen/chitosan porous rack, it is characterized in that the Carbodiimides compound concentrations is the aqueous solution of 0.001～1M.
12. press the preparation method of claims 1 described used in tissue engineering collagen/chitosan porous rack, it is characterized in that the Carbodiimides compound concentrations is the aqueous solution of 0.01～0.3M.
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| WO2006067626A3 (en) * | 2004-12-20 | 2006-08-31 | Carbgraft Ab | Chitosan compositions |
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