CN101530631A - Liver tissue engineering scaffold degradable in vivo and having physiological blood vessel network structure and preparation method thereof - Google Patents
Liver tissue engineering scaffold degradable in vivo and having physiological blood vessel network structure and preparation method thereof Download PDFInfo
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- CN101530631A CN101530631A CN200910061684A CN200910061684A CN101530631A CN 101530631 A CN101530631 A CN 101530631A CN 200910061684 A CN200910061684 A CN 200910061684A CN 200910061684 A CN200910061684 A CN 200910061684A CN 101530631 A CN101530631 A CN 101530631A
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Abstract
A liver tissue engineering scaffold degradable in vivo and having physiological blood vessel network structure and a preparation method thereof are provided, wherein the scaffold is made of galactosylated chitosan, galactosylated or galactose-modified degradable macromolecular materials. The scaffold is a porous sponge body and has a physiological blood vessel network structure therein. The preparation method comprises the following steps: selecting degradable aliphatic polyester materials to obtain a physiological blood vessel tree model by molding blood vessel; then preparing degradable liver tissue engineering scaffold having physiological blood vessel network structure by a combined method of infiltration forming and lyophilization/filtering technology. The scaffold can cause the vascular endothelial cell and liver cell to grow well in pores of tube in blood vessel network and porous sponge body, which is good for the forming of new blood vessel and demand of the cell number in vivo implanted by the scaffold. The inventive liver tissue engineering scaffold has a good biocompatibility, certain mechanical strength and toughness. The preparation technology is simple and easy to operate.
Description
Technical field
The present invention relates to a kind of liver tissue engineering scaffold degradable in vivo that is used for inside and outside hepatocyte or hepatocyte, vascular endothelial cell Mixed culture and preparation method thereof, belong to technical field of bioengineering with liver physiology shape blood vessel network structure.
Background technology
Liver plays very important effect in human body, because the complexity and the irreplaceability of its function, for whole latter stage hepatopathy, how to find that a kind of the substitute of complete liver function is arranged is the difficult point and the focus of medical circle always.At present, more sure method is liver transplantation in whole latter stage hepatopathy, but liver transplantation exists the donor anxiety, operating difficulty is big, beneficiaries are few, expense is high and problem such as rejection, thereby has limited the liver transplantation technology in Clinical Application; And the more methods such as artificial liver of usefulness also exist and can not substitute the liver function of losing merit fully, and exist expense too high, device resource shortage and may cause defective such as blood propagation disease.Thereby the importance and the urgency of artificial organ and tissue regeneration research are particularly important and outstanding.
The essence that engineered organ makes up is to be the assembling of the live body at center with the cell, and constructing primitive is cell, support, signal and active substance.Hepatic tissue organ engineering is compound at the external good degradation material of biocompatibility and the supporting structure and hepatocyte and other cells of utilizing, pass through complex fabrication process, make up a organ, and the ultimate purpose of research is to finish the liver organization organ to rebuild and injury repairing with liver major part or repertoire.In order to realize this goal, must on external support, cultivate the hepatocyte aggregation of the liver function of can or acting normally in a large number, blood confession and long period survival are arranged and have part biological and learn active liver organization, guarantee that it is in survival that implants and propagation, but in its research, still there are many difficult problems to need to solve so far, wherein provide nutrition and gas exchange space for formation and the cell of accelerating blood vessel, promote and support to grow, required liver tissue engineering scaffold and the preparation method with degradable in vivo of blood vessel network structure of the formation of aggregation is one of problem of liver regeneration research in the tissue engineering.
Be generally used for the support of liver tissue engineering research, its internal structure mostly is a kind of simple loose structure.In recent years, researcher constantly is established and develops for the method that preparation contains the vasoganglion structure stand both at home and abroad, simulates moulding etc. as little manufacturing and 3 D-printing technology, laser fast shaping and utilization computer.Satoshi etc. utilize to xerox the slabstone technology, and on the template that silicon and heat resistant glass are made, etching forms the complete vasoganglion with ramose blood vessels at different levels and blood capillary; Usefulness three dimensional printing technology such as Stephen produce and include netted continuous channelization PLGA support; Junji Fukuda etc. makes the porous support of little cylindrical recess form with little manufacturing and little printing technique with collagen/Polyethylene Glycol complex timbering material, and people such as professor Xu Yingxin carries out the liver tissue engineering vascularization on the liver tissue engineering scaffold with the preparation of methods such as laser fast shaping, computer simulation moulding research also shows: the support of pipe network structure is beneficial to the vascularization of hepatic tissue.So in hepatic tissue regeneration research, the tissue engineering bracket that has blood vessel network structure in the support is most important.But carry out the preparation of liver tissue engineering scaffold with said method, except that needing expensive miniature process equipment, complex forming technology and processing cost height, cause easily in the technological forming course of processing of complexity that stent size is restricted, demoulding difficulty, also require to have limited the kind of material because of preparation condition, can control pipeline or hole form easily as ultraviolet curing process, but the exploitation that possesses the photosensitive resin material of good biocompatibility and degradation characteristic lags behind, and makes the characteristic error of rami hepatici frame of molding bigger; Though a series of problems such as but fusion sediment method straight forming Biodegradable material exists pipe diameter bigger, and pipeline form controllability is relatively poor all can't reach the structure of duplicating vasoganglion in the liver fully.Consider as the design angle that has physiology shape blood vessel network structure in from support, and solve existing problem in the above-mentioned preparation pipe network support, prepare the liver tissue engineering scaffold of degradable in vivo with liver physiology shape blood vessel network structure, can promote the vascularization of hepatic tissue and be beneficial to the formation of hepatic tissue, be that people thirst for the technical problem that solves always.
Summary of the invention
The objective of the invention is to propose a kind of liver tissue engineering scaffold degradable in vivo and preparation method thereof with physiological blood vessel network structure.
For achieving the above object, the liver tissue engineering scaffold degradable in vivo with liver physiology shape blood vessel network structure provided by the invention is made by the degradable high polymer material that galactosylation chitosan, galactosylation or galactose are modified.
The substitution value of galactose is 10%-30% in the described liver tissue engineering scaffold degradable in vivo material with liver physiology shape blood vessel network structure.
Liver tissue engineering scaffold degradable in vivo with liver physiology shape blood vessel network structure provided by the invention is the mandruka body, has the structure of liver physiology shape blood vessel network in the support.The mould that contoured cradle is selected according to the size of blood vessel casting model.
The associated methods preparation of casting mold, infiltration forming and lyophilization/leaching technology is adopted in preparation with liver tissue engineering scaffold degradable in vivo of liver physiology shape blood vessel network structure of the present invention, and method comprises the following steps:
(1) carry out the liver vessel casting mold with degradable type aliphatic polyester series cast agent: casting method has two kinds, and a kind of is to carry out living animal liver vessel casting mold, solidifies the back at cast agent and obtains the liver vessel tree-model by removing tissue and cleaning; Another kind is in the vascular tree mould, injects degradable type cast agent, treats that cast agent solidifies the back and takes out the vascular tree model; Its vascular tree mold materials is paraffin, Gypsum Fibrosum, politef or rustless steel, described degradable type aliphatic polyester series cast agent comprises: any one or two kinds of and organic solvent are the cast agent that acetone, ethyl acetate, chloroform or 1,4-dioxane are formed in polylactic acid, polyglycolic acid, the poly-second lactone;
(2) the vascular tree model is put into the container that matches with it, and add porogen or do not add porogen according to the size in the required aperture of liver tissue engineering scaffold spongy body, described porogen is the microgranule of aliphatic polyester series polylactic acid, polyglycolic acid or poly-second lactone, and particle size is 50 μ m～800 μ m;
(3) in the container that has added porogen, use the method adding galactosylation chitosan solution of infiltration forming or the degradable high polymer material solution of galactosylation or modification, the degradable high polymer material solution that in the container that does not add porogen, directly adds galactosylation chitosan solution or galactosylation or modification, carry out freezing then, cryogenic temperature is-10～-196 ℃, treat to carry out lyophilization again behind the solution cryofixation, promptly get the embryo of liver tissue engineering scaffold;
(4) above-mentioned support embryo is immersed organic solvent-acetone, ethyl acetate, chloroform or 1, in the 4-dioxane, treat the vascular tree model of aliphatic polyester and porogen dissolves fully and all gone out by drop after, through distilled water or normal saline flushing postlyophilization, promptly obtain having the liver tissue engineering scaffold semifinished product of liver physiology shape blood vessel network structure;
(5) above-mentioned semifinished product support is put into the anticoagulation medicine heparin and soak, in acetone concentration solution from high to low, carry out polycondensation then, carry out lyophilization again, promptly get liver tissue engineering scaffold product with liver physiology shape blood vessel network structure.In described step 3, perhaps directly anticoagulation medicine heparin, hepatocyte growth factor (HGF) or vascular endothelial cell growth factor (VEGF) are compound in the degradable high polymer material solution of galactosylation chitosan solution or galactosylation or modification.
Advantage of the present invention
Because the degradable macromolecule biomaterial that the galactosylation chitosan that the present invention uses, galactosylation or galactose are modified, galactose in the material is the ligands specific of the asialoglycoprotein receptor (ASGPR) on hepatocyte plasma membrane surface, so it can improve hepatocyte sticking on spongy body, and physiology shape blood vessel network structure in the liver that this support had, can be formation and the hepatocellular survival of accelerating blood vessel nutrition and gas exchange space are provided, promoting and supporting the formation of growth, aggregation and implant becomes possibility.The preparation method of this support is simple, processing ease, need not complicated former and with low cost.
The specific embodiment
Embodiment 1
Preparation with liver tissue engineering scaffold degradable in vivo of rat liver blood vessel network structure, its preparation process is:
1. with acetone solvent, the polylactic acid cast agent solution of preparation 0.05g/ml and 0.1g/ml concentration, rat is carried out live body liver vessel casting mold, the earlier low back of cast agent concentration is high during casting mold, and after-teeming 2 times, downcuts liver then and places room temperature and be placed in mass concentration 25% hydrochloric acid solution corrosion in 2 days 3 days, slowly wash with flowing water, remove cell and connective tissue, soaked 1 day with suds then, can obtain milky rat liver vascular tree model;
2. the vascular tree model is put into the container that matches with it, directly inject the galactosylation chitosan solution, and after inserting 70 ℃ of freezing 24h immediately, carry out lyophilization again and obtain the support blank;
3. above-mentioned support blank is soaked in the acetone, and stirs the stripping of accelerating the vascular tree model gently, change 1-2 no milky polylactic acid in the support spongy body of soak in the immersion, promptly get the support spongy body;
4. after above-mentioned support spongy body being put into distilled water and washing gently, obtain to have the liver tissue engineering scaffold degradable in vivo of rat liver blood vessel network structure after freezing, lyophilization, the pore diameter in the support spongy body is about 50-200 μ m.
Embodiment 2
Preparation with liver tissue engineering scaffold degradable in vivo of rat liver blood vessel network structure, its preparation process is:
1. with acetone solvent, the polylactic acid cast agent solution of preparation 0.05g/ml and 0.1g/ml concentration, rat is carried out live body liver vessel casting mold, the earlier low back of cast agent concentration is high during casting mold, and after-teeming 2 times, downcuts liver then and places room temperature and be placed in mass concentration 25% hydrochloric acid solution corrosion in 2 days 3 days, slowly wash with flowing water, remove cell and connective tissue, soaked 1 day with suds then, can obtain milky rat liver vascular tree model;
2. the vascular tree model is put into the container that matches with it, add an amount of porogen again, porogen is a 200-500 μ m polylactic acid microgranule, slowly injects the galactosylation chitosan solution by seepage flow then, and insert-70 ℃ of freezing 24h immediately, carry out lyophilization again and obtain the support blank;
3. above-mentioned support blank is soaked in the acetone, and stirs the stripping of accelerating vascular tree model and porogen gently.Change 1-2 no milky polylactic acid in the support spongy body of soak in the immersion, promptly get the support spongy body;
4. after above-mentioned support spongy body being put into distilled water and washing gently, obtain to have the liver tissue engineering scaffold degradable in vivo of physiological blood vessel network structure after freezing, lyophilization, the pore diameter in the support spongy body is about 50-500 μ m.
Embodiment 3
Preparation with liver tissue engineering scaffold degradable in vivo of rat liver blood vessel network structure, its preparation process is:
1. with acetone solvent, the polylactic acid cast agent solution of preparation 0.05g/ml and 0.1g/ml concentration, rat is carried out live body liver vessel casting mold, the earlier low back of cast agent concentration is high during casting mold, and after-teeming 2 times, downcuts liver then and places room temperature and be placed in mass concentration 25% hydrochloric acid solution (perhaps 10% sodium hydroxide) corrosion in 2 days 3 days, slowly wash with flowing water, remove cell and connective tissue, soaked 1 day with suds then, can obtain milky rat liver vascular tree model;
2. the vascular tree model is put into the container that matches with it;
3. the porogen of adding 30% obtains mixed solution in the galactosylation chitosan solution, and porogen is a 300-800 μ m polylactic acid microgranule;
4. above-mentioned mixed solution is poured in the container that is placed with the vascular tree model, and inserted-70 ℃ of freezing 24h immediately, carry out lyophilization again and obtain the support blank;
5. above-mentioned support blank is soaked in the acetone, and stirs the stripping of accelerating vascular tree model and porogen gently, change 1-2 no milky polylactic acid in the support spongy body of soak in the immersion, promptly get the support spongy body.
6. after above-mentioned support spongy body being put into distilled water and washing gently, obtain to have the liver tissue engineering scaffold degradable in vivo of physiological blood vessel network structure after freezing, lyophilization, the pore diameter in the support spongy body is about 50-800 μ m.
Claims (5)
1. the liver tissue engineering scaffold degradable in vivo with liver physiology shape blood vessel network structure is characterized in that being made by the degradable high polymer material that galactosylation chitosan, galactosylation or galactose are modified.
2. according to the described liver tissue engineering scaffold degradable in vivo of claim 1, it is characterized in that the substitution value of galactose is 10%-30% in its timbering material with liver physiology shape blood vessel network structure.
3. according to the described liver tissue engineering scaffold degradable in vivo of claim 1, it is characterized in that described support is the mandruka body, have liver physiology shape blood vessel network structure in the support with liver physiology shape blood vessel network structure.
4. the described preparation method with liver tissue engineering scaffold degradable in vivo of liver physiology shape blood vessel network structure of claim 1 is characterized in that, adopts the associated methods of casting mold, infiltration forming and lyophilization/leaching technology, and method step comprises:
(1) carry out the liver vessel casting mold with degradable type aliphatic polyester series cast agent: casting method has two kinds, and a kind of is to carry out living animal liver vessel casting mold, solidifies the back at cast agent and obtains the liver vessel tree-model by removing tissue and cleaning; Another kind is in the vascular tree mould, injects degradable type cast agent, treats that cast agent solidifies the back and takes out the vascular tree model; Its vascular tree mold materials is paraffin, Gypsum Fibrosum, politef or rustless steel, described degradable type aliphatic polyester series cast agent comprises: any one or two kinds of and organic solvent are the cast agent that acetone, ethyl acetate, chloroform or 1,4-dioxane are formed in polylactic acid, polyglycolic acid, the poly-second lactone;
(2) the vascular tree model is put into the container that matches with it, and add porogen or do not add porogen according to the size in the required aperture of liver tissue engineering scaffold spongy body, described porogen is the microgranule of aliphatic polyester series polylactic acid, polyglycolic acid or poly-second lactone, and particle size is 50 μ m～800 μ m;
(3) in the container that has added porogen, use the method adding galactosylation chitosan solution of infiltration forming or the degradable high polymer material solution of galactosylation or modification, the degradable high polymer material solution that in the container that does not add porogen, directly adds galactosylation chitosan solution or galactosylation or modification, carry out freezing then, cryogenic temperature is-10～-196 ℃, treat to carry out lyophilization again behind the solution cryofixation, promptly get the embryo of liver tissue engineering scaffold;
(4) above-mentioned support embryo is immersed organic solvent-acetone, ethyl acetate, chloroform or 1, in the 4-dioxane, treat the vascular tree model of aliphatic polyester and porogen dissolves fully and all gone out by drop after, through distilled water or normal saline flushing postlyophilization, promptly obtain having the liver tissue engineering scaffold semifinished product of liver physiology shape blood vessel network structure;
(5) above-mentioned semifinished product support is put into the anticoagulation medicine heparin and soak, in acetone concentration solution from high to low, carry out polycondensation then, carry out lyophilization again, promptly obtain to have the liver tissue engineering scaffold product of liver physiology shape blood vessel network structure.
5. according to the described preparation method of claim 4 with liver tissue engineering scaffold degradable in vivo of liver physiology shape blood vessel network structure, it is characterized in that, in described step (3), perhaps directly anticoagulation medicine heparin, hepatocyte growth factor and vascular endothelial cell growth factor are compound in the degradable high polymer material solution of galactosylation chitosan solution or galactosylation or modification.
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| CN200910061684A CN101530631A (en) | 2009-04-21 | 2009-04-21 | Liver tissue engineering scaffold degradable in vivo and having physiological blood vessel network structure and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690435A (en) * | 2012-06-19 | 2012-09-26 | 上海大学 | Method for regulating pore structure of water-soluble polymer tissue engineering scaffold by use of polyester template |
| CN105999433A (en) * | 2016-05-11 | 2016-10-12 | 山东省药学科学院 | Polyglycolic acid particle doped polylactic acid-trimethylene carbonate film and preparation method and application thereof |
| CN108588006A (en) * | 2018-05-10 | 2018-09-28 | 华东理工大学 | A kind of biological support and its preparation method and application for liver cell dimensional culture |
| CN110384823A (en) * | 2019-07-19 | 2019-10-29 | 大连医科大学 | Bionical lobuli hepatis and construction method based on silk fibroin bracket |
| CN110859999A (en) * | 2019-10-11 | 2020-03-06 | 南方医科大学 | Construction method of three-dimensional vascular network hydrogel |
| CN115040696A (en) * | 2022-06-07 | 2022-09-13 | 重庆市畜牧科学院 | Artificial blood vessel with low immunogenicity and preparation method thereof |
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2009
- 2009-04-21 CN CN200910061684A patent/CN101530631A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690435A (en) * | 2012-06-19 | 2012-09-26 | 上海大学 | Method for regulating pore structure of water-soluble polymer tissue engineering scaffold by use of polyester template |
| CN105999433A (en) * | 2016-05-11 | 2016-10-12 | 山东省药学科学院 | Polyglycolic acid particle doped polylactic acid-trimethylene carbonate film and preparation method and application thereof |
| CN105999433B (en) * | 2016-05-11 | 2019-03-19 | 山东省药学科学院 | A kind of polylactic acid-trimethylene carbonate film and its preparation method and application adulterating polyglycolic acid particle |
| CN108588006A (en) * | 2018-05-10 | 2018-09-28 | 华东理工大学 | A kind of biological support and its preparation method and application for liver cell dimensional culture |
| CN110384823A (en) * | 2019-07-19 | 2019-10-29 | 大连医科大学 | Bionical lobuli hepatis and construction method based on silk fibroin bracket |
| CN110384823B (en) * | 2019-07-19 | 2021-08-03 | 大连医科大学 | Bionic liver lobule based on silk fibroin scaffold and construction method |
| CN110859999A (en) * | 2019-10-11 | 2020-03-06 | 南方医科大学 | Construction method of three-dimensional vascular network hydrogel |
| CN115040696A (en) * | 2022-06-07 | 2022-09-13 | 重庆市畜牧科学院 | Artificial blood vessel with low immunogenicity and preparation method thereof |
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Application publication date: 20090916 |