CN1748803A - Method for preparing heparin collagen/chitosan porous rack of composite angiogenin - Google Patents
Method for preparing heparin collagen/chitosan porous rack of composite angiogenin Download PDFInfo
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Abstract
The preparation process of porous heparinized collagen/chitosan rack with compounded angiogenin includes the following steps: dissolving ox tendon and chitosan separately in acetic acid solution to compound 0.5-5 % concentration solution, mixing chitosan solution in 5-30 % and ox tendon solution, molding and freeze drying to obtain porous collagen/chitosan rack; vacuum treatment, soaking the porous collagen/chitosan rack inside 2-N-morpholynyl ethane sulfonic acid solution of heparin sodium, treating with 1-ethyl-3-3-(dimethylaminopropyl)-carbonized diimine solution and N-hydroxy batanimide solution, washing, re-freezing to dry and to obtain porous heparinized collagen/chitosan rack; and soaking the porous heparinized collagen/chitosan rack inside angiogenin solution to obtain the porous heparinized collagen/chitosan rack with compounded angiogenin. The prepared rack has proper pore size and porosity and is used as the corium substitute in skin tissue engineering.
Description
Technical field
The present invention relates to the preparation method that a kind of composite vascular generates plain heparinization collagen/chitosan porous rack.
Background technology
The reparation that appears as skin wound of tissue engineering skin and healing provide a brand-new treatment approach, but, present various tissue engineering skin transplanting survival rate is starkly lower than from the body split-thickness skin graft transplants, one of them main cause is that tissue engineering skin is transplanted back vascularization speed than slow from the body split-thickness skin graft, is to improve the key factor of its survival rate so accelerate to knit through engineering approaches skin heart speed.At present, use that to have the somatomedin that promotes angiogenesis function be to promote of tissue engineering skin vascularization than good method.
In numerous somatomedin, angiogenin (angiogenin, ANG) being unique up to now one promotes angiogenesis ability based on it and is found angiogenesis factor with separation and purification, can combine with heparin, it is that a molecular weight of being made up of 123 aminoacid is a 14kDa strand basic protein, it promotes that the angiogenesis ability is very strong, and the amount of nanogram level can work.But the angiogenin half-life in vivo is very short, within several minutes, promptly degrade and lose biological activity, and be exactly the sustained release system that can obtain angiogenin so solve its key in application.
Summary of the invention
The purpose of this invention is to provide the preparation method that a kind of composite vascular generates plain heparinization collagen/chitosan porous rack, can promote its vascularization speed in vivo, thereby improve the transplanting survival rate.
The step of method is as follows:
1) respectively beef tendon collagen, chitosan are mixed with 0.5 ~ 5% solution with the acetic acid solution of 0.5mol/l, beef tendon collagen solution and chitosan solution mix homogeneously inject mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 5～30% ,-20～-50 ℃ of rearmounted freeze dryers of freezing 1～5h;
2) the gained collagen/chitosan porous rack under vacuum 80 ~ 130 ℃ handle 12～48h after, it is dipped in the 2-N-morpholino ethane sulfonic acid solution that contains heparin sodium, the mass ratio of heparin sodium is 4～80: 1 in beef tendon collagen and the solution, soak 0.5～2h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor then and handle 12～48h, wherein the molar concentration rate of 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor is 0.5～5: 1, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack;
3) gained heparinization collagen/chitosan porous rack is dipped in the angiogenesis cellulose solution, and 4 ~ 25 ℃ are soaked 2～24h.
Chitosan solution content (by weight) is 5～30% in beef tendon collagen and the chitosan mixed solution.The mass ratio of heparin sodium is 4～10: 1 in beef tendon collagen and the solution.1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution concentration is 20～80mmol/l.The concentration ratio of 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor is 1～2.5: 1.The soaking temperature of heparinization collagen/chitosan porous rack and angiogenesis cellulose solution is 4～16 ℃, and soak time is 8～10h.
The heparinization collagen/chitosan porous rack of the present invention's preparation has suitable aperture and porosity, is fit to the dermal substitute of skin tissue engineering.Adopt angiogenin can promote the vascularization of dermal substitute fast, improve the transplanting survival rate.The control delivery method that the present invention prepares angiogenin is extremely simple, effective, and the influence factor is few, more easily promotes.
Description of drawings
Fig. 1 (a) is scanning electron microscope (SEM) photo of not heparinization collagen/chitosan porous rack;
Fig. 1 (b) is scanning electron microscope (SEM) the photo partial enlarged drawing of not heparinization collagen/chitosan porous rack;
Fig. 1 (c) is scanning electron microscope (SEM) photo of heparinization collagen/chitosan porous rack;
Fig. 1 (d) is scanning electron microscope (SEM) the photo partial enlarged drawing of heparinization collagen/chitosan porous rack;
Fig. 2 (a) is that different blood vessel generates the influence figure of plain concentration to support angiogenin binding capacity;
Fig. 2 (b) is the influence figure of heparinization to support angiogenin combination rate;
Fig. 3 is the influence figures of different chitosan solution content to heparinization collagen/chitosan porous rack angiogenin combination rate;
Fig. 4 be in different beef tendon collagens and the solution heparin sodium mass ratio to the figure that influences of heparinization collagen/chitosan porous rack angiogenin combination rate;
Fig. 5 is the influence figure of different 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution concentration to heparinization collagen/chitosan porous rack angiogenin combination rate;
Fig. 6 is the figure that influences that the molar concentration of different 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides (EDC) solution and N-maloyl imines (NHS) solution is compared heparinization collagen/chitosan porous rack angiogenin combination rate;
The different soaking temperatures of Fig. 7 are to the figure that influences of heparinization collagen/chitosan porous rack angiogenin combination rate;
Fig. 8 is the influence figures of different soak times to heparinization collagen/chitosan porous rack angiogenin combination rate;
Fig. 9 is the release in vitro behavior figure of angiogenin;
Figure 10 (a) is that composite vascular generates the tissue slice figure after 1 week of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt;
Figure 10 (b) is that composite vascular does not generate the tissue slice figure after 1 week of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt;
Figure 10 (c) is that composite vascular generates the tissue slice figure after 2 weeks of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt;
Figure 10 (d) is that composite vascular does not generate the tissue slice figure after 2 weeks of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt; Figure 10 (e) is a new vessels counting diagram in the support.
The specific embodiment
Embodiment 1: heparinization is to the influence of collagen/chitosan porous rack micro structure
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are injected mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Fig. 1 a, Fig. 1 c are respectively not scanning electron microscope (SEM) photo of heparinization and heparinization collagen/chitosan porous rack, and Fig. 1 b, Fig. 1 d are respectively the partial enlarged drawing of Fig. 1 a and Fig. 1 c.
Embodiment 2: heparinization is to the influence of angiogenin combination rate
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are injected mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in the 0.25ml angiogenesis cellulose solution, is divided into six groups by angiogenin concentration 0,10,20,40,80 and 160ng/ml; Similarity condition is established six groups, puts into not the heparinization support and organizes in contrast.4 ℃ of immersions were taken out support after 9 hours, and the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain support.The result shows, along with the increase of angiogenin concentration, and heparinization, the not all corresponding increase of the angiogenin binding capacity of heparinization support, concentration and binding capacity are linear; Collagen/chitosan porous rack and angiogenin combination rate are very low, only be 1.7%, and heparinization have significantly improved the binding capacity of the angiogenin of collagen/chitosan support, and its combination rate reaches 36.5%.Plain concentration is to the figure that influences of support angiogenin binding capacity for different blood vessel generates for Fig. 2 (a), and Fig. 2 (b) is the influence figure of heparinization to support angiogenin combination rate.
Embodiment 3: different chitosan solution content are to the influence of heparinization collagen/chitosan porous rack angiogenin combination rate
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are obtained chitosan swelling solution content (by weight) and be respectively 5%, 10%, 20%, 30% mixed liquor, lyophilizing obtains collagen/chitosan porous rack in-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (40ng/ml) solution, 4 ℃ of immersions were taken out support after 9 hours, the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.Fig. 3 is the influence figures of different chitosan solution content to heparinization collagen/chitosan porous rack angiogenin combination rate.
Embodiment 4: the heparin sodium mass ratio is to the influence of heparinization collagen/chitosan porous rack angiogenin combination rate in different beef tendon collagens and the solution
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, beef tendon collagen solution and chitosan solution mix homogeneously inject mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), wherein the mass ratio of heparin sodium is respectively 4,7,10,20,40 and 80: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (40ng/ml) solution, 4 ℃ of immersions were taken out support after 9 hours, the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.Fig. 4 be in different beef tendon collagens and the solution heparin sodium mass ratio to the figure that influences of heparinization collagen/chitosan porous rack angiogenin combination rate.
Embodiment 5: different 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution concentration is to the influence of heparinization collagen/chitosan porous rack angiogenin combination rate
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, beef tendon collagen solution and chitosan solution mix homogeneously inject mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor then and handle 24h, wherein the concentration of 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution is respectively 5,10,20,40 and 80mmol/l, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (40ng/ml) solution, 4 ℃ of immersions were taken out support after 9 hours, the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.Fig. 5 is the influence figure of different 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution concentration to heparinization collagen/chitosan porous rack angiogenin combination rate.
Embodiment 6: the influence of the molar concentration comparison heparinization collagen/chitosan porous rack angiogenin combination rate of different 1-ethyl-3-3-(dimethyl amine propyl group)～carbodiimides (EDC) solution and N-maloyl imines (NHS) solution
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, beef tendon collagen solution and chitosan solution mix homogeneously inject mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor then and handle 24h, wherein the concentration of 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution is 40mmol/l, the molar concentration rate of 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor is respectively 0.5,1,1.5,2,2.5,5: 1, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (40ng/ml) solution, 4 ℃ of immersions were taken out support after 9 hours, the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.Fig. 6 is the figure that influences that the molar concentration of different 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides (EDC) solution and N-maloyl imines (NHS) solution is compared heparinization collagen/chitosan porous rack angiogenin combination rate.
Embodiment 7: different soaking temperatures are to the influence of heparinization collagen/chitosan porous rack angiogenin combination rate
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are injected mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (40ng/ml) solution, after soaking 9 hours under 4 ℃, 8 ℃, 16 ℃, 25 ℃, take out support respectively, the PBS rinsing is 2 times under the room temperature, each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.Fig. 7 is the influence figures of different soaking temperatures to heparinization collagen/chitosan porous rack angiogenin combination rate.
Embodiment 8: different soak times are to the influence of heparinization collagen/chitosan porous rack angiogenin combination rate
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are injected mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (40ng/ml) solution, soak respectively under 4 ℃ 2,4,6,8,10,12 and 14h after take out support, the PBS rinsing is 2 times under the room temperature, each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.Fig. 8 is the influence figures of different soak times to heparinization collagen/chitosan porous rack angiogenin combination rate.
Embodiment 9: the release in vitro behavior of angiogenin
The method that combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are injected mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add l-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.25mg) is dipped in 0.25ml angiogenin (160ng/ml) solution, take out support after soaking 9h respectively under 4 ℃, the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.The angiogenin binding capacity is the 14.6ng/ sheet; Obtain composite vascular with quadrat method and generate plain not heparinization collagen/chitosan porous rack, the binding capacity of angiogenin is the 0.7ng/ sheet.Composite vascular is generated two kinds of plain supports put into 0.5ml release liquid (the CS-C endotheliocyte culture medium that contains 10% HAS, 100U/ml penicillin, 100 μ g/ml respectively, pH7.2～7.4) in, place 37 ℃ of constant temperature cell culture incubators, regularly take out release liquid and measure the angiogenin burst size.The angiogenin release that composite vascular generates plain heparinization collagen/chitosan porous rack is continual and steady type, sees Fig. 9.
Embodiment 10: the method that the plain interior vascularization evaluation of heparinization collagen/chitosan porous rack body of composite vascular generation combines with sour extracting with enzymolysis is extracted from beef tendon and is obtained beef tendon collagen.Respectively collagen, chitosan are mixed with 0.5% solution with the acetic acid solution of 0.5mol/l, then beef tendon collagen solution and chitosan solution mix homogeneously are injected mould, chitosan solution content (by weight) is that lyophilizing obtains collagen/chitosan porous rack in 10% ,-20 ℃ of rearmounted freeze dryers of freezing 2h; The gained collagen/chitosan porous rack under vacuum 105 ℃ handle 24h after, it is dipped in 2-N-morpholino ethane sulfonic acid (50mmol/l) solution that contains heparin sodium (available from U.S. SIGMA company), the mass ratio of heparin sodium is 10: 1 in beef tendon collagen and the solution, soak 1h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution (40mmol/l) and N-maloyl imide liquor (20mmol/l) then and handle 24h, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack; Heparinization collagen/chitosan porous rack (0.2mg) is dipped in 3ml angiogenin (320ng/ml) solution, 4 ℃ of immersions were taken out support after 9 hours, the PBS rinsing is 2 times under the room temperature, and each 10min obtains composite vascular and generates plain heparinization collagen/chitosan porous rack.This support is imbedded under the rabbit ear butt, same heeling-in not composite vascular generates plain heparinization support in contrast, make tissue slice observes respectively at drawing materials in the 1st, 2 weeks after the heeling-in, rabbit ear back of the body arterial perfusion india ink before drawing materials, tissue slice is counted new vessels number (unit: individual/H, H represents 100 times of visuals field) under 100 times of visuals field.In 1 week after the heeling-in, composite vascular generates the visible more new vessels of plain support and grows into, and vessel density (10.1 ± 4.2) is individual/H, but vascular tissue is not seen in the deep; Matched group is only at support and rabbit ear tissue boundary visible vessels tissue.In 2 weeks of postoperative, experimental group support new vessels has reached the support deep, but fully vascularization, and vessel density (38.2 ± 6.8) is individual/H, than the 1st week obviously increasing (P＜0.01); Matched group support new vessels concentrates on periphery, and blood vessel structure is not seen in the deep, density be (22.6 ± 6.9) individual/H, significantly less than with the time point experimental group.Figure 10 (a) is that composite vascular generates the tissue slice figure after 1 week of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt; Figure 10 (b) is that composite vascular does not generate the tissue slice figure after 1 week of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt; Figure 10 (c) is that composite vascular generates the tissue slice figure after 2 weeks of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt; Figure 10 (d) is that composite vascular does not generate the tissue slice figure after 2 weeks of heeling-in under the plain heparinization collagen/chitosan porous rack rabbit ear butt; Figure 10 (e) is a new vessels counting diagram in the support.
Claims (7)
1. a composite vascular generates the preparation method of plain heparinization collagen/chitosan porous rack, it is characterized in that the step of method is as follows:
1) respectively beef tendon collagen, chitosan are mixed with 0.5 ~ 5% solution with the acetic acid solution of 0.5mol/l, beef tendon collagen solution and chitosan solution mix homogeneously inject mould, the sub-content of chitosan solution weight percent is that lyophilizing obtains collagen/chitosan porous rack in 5～30% ,-20～-50 ℃ of rearmounted freeze dryers of freezing 1～5h;
2) the gained collagen/chitosan porous rack under vacuum 80 ~ 130 ℃ handle 12～48h after, it is dipped in the 2-N-morpholino ethane sulfonic acid solution that contains heparin sodium, the mass ratio of heparin sodium is 4～80: 1 in beef tendon collagen and the solution, soak 0.5～2h, add 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor then and handle 12～48h, wherein the molar concentration rate of 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor is 0.5～5: 1, clean, freezing once more, lyophilizing obtains the heparinization collagen/chitosan porous rack;
3) gained heparinization collagen/chitosan porous rack is dipped in the angiogenesis cellulose solution, and 4 ~ 25 ℃ are soaked 2～24h.
2. a kind of composite vascular according to claim 1 generates the preparation method of plain heparinization collagen/chitosan porous rack, it is characterized in that described beef tendon collagen and chitosan mixed solution, and the sub-content of chitosan solution weight percent is 5～30%.
3. a kind of composite vascular according to claim 1 generates the preparation method of plain heparinization collagen/chitosan porous rack, and the mass ratio that it is characterized in that heparin sodium in described beef tendon collagen and the solution is 4～10: 1.
4. a kind of composite vascular according to claim 1 generates the preparation method of plain heparinization collagen/chitosan porous rack, it is characterized in that described 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution concentration is 20～80mmol/l.
5. a kind of composite vascular according to claim 1 generates the preparation method of plain heparinization collagen/chitosan porous rack, it is characterized in that the concentration ratio of described 1-ethyl-3-3-(dimethyl amine propyl group)-carbodiimides solution and N-maloyl imide liquor is 1～2.5: 1.
6. a kind of composite vascular according to claim 1 generates the preparation method of plain heparinization collagen/chitosan porous rack, and the soaking temperature that it is characterized in that described heparinization collagen/chitosan porous rack and angiogenesis cellulose solution is 4～16 ℃.
7. a kind of composite vascular according to claim 1 generates the preparation method of plain heparinization collagen/chitosan porous rack, and the soak time that it is characterized in that step 3) is 8～10h.
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| CN106905443B (en) * | 2017-02-20 | 2019-03-01 | 江苏省农业科学院 | The preparation method and applications of guanidinated chitosan |
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