CN1762311A - Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction - Google Patents
Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction Download PDFInfo
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Abstract
The invention relates to the method for preparing the biological vessel with clove used for the restoring and rebuilding of the cardiovascular system tissue, especially to the method with the material got from foreign biology tissue. The preparing method of the biological vessel with clove for the restoring and rebuilding of the arteria pulmonalis vessel comprising the following steps: 1) getting the cow jugular vein, poaching and sterilizing; 2) cross bonding the base material of the cow jugular vein through the light oxidation reaction induced by dye; 3) sterilizing and storing; with the characteristic to be the treatment of the cow jugular vein in step 1) with cell-removing agent before step 2). The biological vessel with clove prepared by the method in this invention possesses good biological and physical property, calcification-resistant property, low immunogen and non cell toxicity, which benefits the growth of the host cell.
Description
Technical field
The present invention relates to preparation and be used for the method, particularly source of drawing material of porcine cardiovascular conduit of cardiovascular system tissue reparation or reconstruction in the method for heterogeneity biological organization.
Technical background
Be connected hypogenetic complicated congenital heart disease such as truncus arteriosus communis in the right ventricle with pulmonary artery, pulmonary atresia, double outlet of right ventricle, the surgical intervention of modified TGA etc. needs the applying biological valved conduit.There is not valved conduit, this class disease therapeuticing effect extreme difference.Since the sixties in last century, adopted band lobe aorta of the same race or pulmonary artery pipeline both at home and abroad, and obtained good result.But valved conduit of the same race source difficulty is particularly suitable for the infant operation still less, and, there is calcification at a specified future date, problem such as durability is not good.Develop porcine pulmonary artery or aorta valved conduit the eighties abroad, but clinical application effect is not good enough, calcification at a specified future date is serious, loses merit and decays poor durability.Beginning 90 years last century carries out the research of bovine jugular vein valved conduit abroad and is applied to clinical.The bovine jugular vein valved conduit has draws materials conveniently, and the natural valve that has is similar to people's valve of pulmonary trunk structure, and anti-reflux performance is strong, and the blood vessel diameter span scope is big, is more suitable in clinical infant case.At present in the world, unique bovine jugular vein valved conduit product is called ' Congtegra ' for the commodity that Medtronic company produces.This product system adopts conventional biological tissue cross-linking agent glutaraldehyde fixedly to be prepared from.This product is used in states such as America and Europes, and near and mid-terms clinical effectiveness report is the result differ.The reason of poor effect is mainly tissue calcification, anastomotic stricture and thrombosis etc.And these reasons are main relevant with technology of preparing.Glutaraldehyde cross-linking is handled biological tissue and is had following defective: 1) cytotoxic effect, and the medium-term and long-term residual toxic effect of glutaraldehyde pair cell of tissue after crosslinked, host cell can not be grown in tissue.2) reasons such as existence of aldehyde radical cause organizing easy calcification after crosslinked, and calcification is one of major reason of decaying of valved conduit.3) immunogenicity is eliminated not exclusively, is one of reason of calcification, also is to cause one of inflammatory reaction mechanism.4) residual cell and chip are one of main sites of tissue calcification, also are immunogenic important sources.Fig. 1 is a sample drawing (A) of handling the valved conduit that obtains with traditional glutaraldehyde method, and reaches interior change after 1 year (B) of implantation dog body; Change (B) after 1 year in bovine jugular vein valve (A) that Fig. 2 glutaraldehyde is handled and the implantation dog body.
Improving the Processing of Preparation technology is to improve the important step of bovine jugular vein valved conduit performance.
The photooxidation of dyestuff mediation is crosslinked a kind of non-the mixing property technology of heterogeneity biological organization.Be to make tissue collagen crosslinked by photochemical reaction, no cytotoxicity.For different tissues, the photooxidation fixing means is variant on some details, but the macromethod unanimity promptly is dissolved with in the solution of dyestuff wanting fixed material to immerse, and carries out light radiation.Used dyestuff has following several: methylene blue, methylene green, rose bengal, riboflavin, proflavine, fluorescein, five pyridoxal 5-phosphates, Yihong etc.Wherein using maximum is methylene blue, and the working concentration of dyestuff is 0.01%～0.1%.The solution that uses can be low ionic strength buffer liquid, high ionic strength buffers liquid, phosphate buffered saline(PBS) or tissue buffer solution.The pH value 6.8～8.6 of solution, osmotic pressure are about 300mmol/kg, 0 ℃～25 ℃ of solution temperatures, and the best is about 10 ℃, can avoid the too high degeneration that causes histone of temperature.According to the difference of fixed material, the intensity and the time of light radiation are different, generally are the bulbs with 1～2 150～500W, are placed on the height from sample 2～12cm; The time of illumination, there were a comprehensive index-lumen-hour intensity of illumination and time from a few minutes to 160h, general 100～20000 lumen-hours.
Be not used for handling the method for bovine jugular vein valved conduit by the photooxidation crosslinking technological of disclosed dyestuff mediation in the prior art.The applicant handles the bovine jugular vein valved conduit with this technology, and its result remains undesirable.
On the other hand, go cell technology in tissue engineering field extensive use.Removing the extracellular matrix that keeps behind the cell, is a kind of natural supporting structure that host cell adheres to, grows and moves that helps.The abundant removal of cell component has reduced immunogenicity, has also eliminated this possible calcification site of the residual nuclear of cell, has improved the calcification performance.Bibliographical information goes the method for cell to have two kinds: (1) detergent takes off cell: detergent is the water-soluble lipid of a class, and it has hydrophilic segment and hydrophobic part, and therefore energy cracking adipose membrane dissolves antigen, removes immune complex.Detergent is divided into: 1. ion-type: electrically charged head (+or-), as sodium lauryl sulphate (SDS), sodium cholate, NaTDC etc.Its shortcoming is to make the degeneration of protein height, and protein is separated with monomeric form.2. nonionic: be with nonpolar head, as Triton X-100, Triton-X114, hot glucoside, Tween20 etc.A little less than it disturbs to the interaction between protein and protein, to the effect of protein denaturation also a little less than.3. amphoteric: have+and-the electric charge head, as CHAPS, Zwitterget etc.(2) digestive enzyme takes off cell: adopt trypsin, deoxyribonuclease and ribonuclease as taking off the cell agent.These two kinds are taken off Cell sap, can freely select according to different tissues and clinical practice situation.The general associating using method that adopts.
Summary of the invention
The present invention is the porcine cardiovascular conduit that is provided for pulmonary artery reparation or reconstruction, adopts and goes the photooxidation crosslinking technological of cell technology and dyestuff mediation to handle the bovine jugular vein valved conduit.
The preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction provided by the invention comprises the steps: 1) obtain bovine jugular vein, and rinsing, sterilization; 2) adopt the photooxidation reaction of dyestuff mediation to make bovine jugular vein substrate crosslinked; 3) sterilization and preservation; It is characterized in that before the 2nd step, spending earlier the cell agent and handle the bovine jugular vein that the 1st step obtained.
Key of the present invention is to select the suitable protein cross method of going the cell method and adopting non-glutaraldehyde.
The photooxidation crosslinking technological Combined Treatment bovine jugular vein valved conduit of cell technology and dyestuff mediation is removed in employing, can utilize both advantages, makes the valved conduit of preparation reach good performance.As going cell technology, its effect is that cell component in the bovine jugular vein histiocyte such as nucleus etc. are fully removed, and eliminates the calcification site thereby reach, and reduces immunogenic purpose.And the photooxidation The Application of Technology of dyestuff mediation is to make the protein in the tissue full cross-linked, and a stable framing structure is provided.
Bovine jugular vein picks up from this bamboo top and Qinghai yak, and body weight is 100～600 kilograms, health, and it is qualified to quarantine.Bovine jugular vein has many group valves usually, and every group of valve is generally SANYE or two leaves, and small part is four leaves.Lobe leaf development varying degree has the bovine jugular vein of 20-30% to have growth and anti-reflux well behaved valve approximately.The lobe leaf is poor, is similar to people's pulmonary artery.Lobe hole place blood vessel wall is thin, easy damaged during pruning.The statistical result of same bovine jugular vein valve group distance is 6.4 ± 2.6cm.Bovine jugular vein has many little vessel branch openings usually, needs ligation during preparation.Bovine jugular vein adventitia fibrous connective tissue is abundant, and is unclear with middle film boundary.Jugular valve is grown not have obviously and is distinguished between the difference cattle system.Blood vessel diameter is 8～22mm, removes fascia tissue under aseptic condition, detects valve developmental condition and anti-reflux performance.Valve is grown and anti-reflux well behaved blood vessel immerses 0.1% bromo geramine 30min, PBS rinsing.
According to prior art, the detergent that is used to cell has multiple, comprises ion-type, nonionic and amphoteric.The present invention selects non-ionic detergent for use, and for example bent that leads to 100.
The present invention adopts the rapid means of multistep in going the step of cell.Except that adopting non-ionic detergent, also to unite and adopt digestive enzyme or/and nuclease, used digestive enzyme is a trypsin, nuclease is deoxyribonuclease and ribonuclease.
Therefore, the present invention further provides method that cell handles is to unite the detergent that adopts nonionic and digestive enzyme or/and the method for nuclease processing; The concentration of detergent is 0.25-1%, and digestive enzyme is a trypsin; Nuclease is deoxyribonuclease and ribonuclease, and concentration is 0.025～0.05% trypsin when digestive enzyme is trypsin; When digestive enzyme was deoxyribonuclease and ribonuclease, its concentration was respectively 10-40unit/ml and 0.1-0.4mg/ml.
By disclosed method in the prior art, when removing cell, also has certain density EDTA in the solution with trypsin.
It is above-mentioned that various to go the processing time of cell agent also be a very important parameters.The preferred stain release agent processing time is 24--48 hour among the present invention, and the trypsin treatment time is 30--120 minute, and deoxyribonuclease and ribonuclease mixing post processing time are 24-72 hour.
Those logical 100 concentration of optimum selected songs are 0.05%, and the processing time is 48 hours, and digestive enzyme is a trypsin, and its concentration is 0.025-0.125%, and the processing time is 30--60 minute; And deoxyribonuclease and ribonuclease, its concentration is respectively 20unit/ml and 0.2mg/ml, and the processing time is 24 hours.
For photooxidation reaction, specifically selected methylene blue among the present invention for use, and at pH:7.6; Under the Osm:320mosm condition, the unsettled irradiation 20～48h of 150～500W electric filament lamp.
Preferably in methylene blue solution, PBS (pH:7.6; Osm:320mosm) soaked 2-4 hour under the condition.
When carrying out photooxidation reaction, porcine cardiovascular conduit is in and is not higher than under 25 ℃ of temperature conditions.For example can not be higher than 25 ℃ with the temperature of the processed porcine cardiovascular conduit of the method control of water-bath.
The preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction provided by the invention can obtain to have good biophysics performance, and the calcification performance is strong, and immunogenicity is low, and no cytotoxicity helps the porcine cardiovascular conduit that host cell is grown.
Description of drawings
Fig. 1 is bovine jugular vein valved conduit (A) and interior change after 1 year (B) of implantation dog body that glutaraldehyde is handled;
Fig. 2 is bovine jugular vein valve (A) and interior change after 1 year (B) of implantation dog body that glutaraldehyde is handled; Wherein arrow is indicated valve structure;
After Fig. 3 handled with method of the present invention, bovine jugular vein blood vessel (A) and valve (B) tissue morphology changed (HE dye X20);
After Fig. 4 handles with method of the present invention, change (B) after 1 year in bovine jugular vein valved conduit (A) and the implantation dog body, wherein arrow indication position of valve;
After Fig. 5 handles with method of the present invention, change (B) after 1 year in bovine jugular vein valve (A) and the implantation dog body, wherein arrow indication position of valve;
After Fig. 6 handles with method of the present invention, vascular tissue's endothelioid cells regeneration figure (HE dyeing (X100) arrow shows endothelioid cells) after a year in the bovine jugular vein implantation dog body;
The bovine jugular vein histone of Fig. 7 different technologies Processing of Preparation extracts result of the test, F wherein: flesh tissue, P: tissue is handled in photooxidation, GA: glutaraldehyde is handled tissue, DE: simple de-cellular system, DP: go cell to handle tissue in conjunction with photooxidation, DGA: go cell to handle tissue, M:marker in conjunction with glutaraldehyde.12,24,36 and 48 are the photooxidation processing time;
Fig. 8 utilizes the immunogenicity result of the BJVC of Western blotting technology for detection distinct methods Processing of Preparation, wherein F: fresh group, and GA: glutaraldehyde group, P: photooxidation group, DP: go cell in conjunction with the photooxidation group;
Fig. 9 Human umbilical vein endothelial cells go cell in conjunction with the bovine jugular vein blood vessel sheet of photooxidation technology preparation on plantation back Electronic Speculum result (SEM_x 1200. arrows let others have a look at huve cell).
Specific embodiment
Embodiment 1
1) obtain bovine jugular vein from the slaughterhouse, the cattle kind can be this bamboo top, and body weight is 100～600 kilograms, health, and it is qualified to quarantine.Blood vessel diameter is 8～22mm, removes fascia tissue under aseptic condition, detects valve developmental condition and anti-reflux performance.Valve is grown and anti-reflux well behaved blood vessel immerses 0.1% bromo geramine 30min, PBS rinsing.
2) go cell to handle:
Divide ten groups at random according to table 1.Adopt variable concentrations, different action time, difference to go the cell agent by following method combined treatment vascular tissue.Observe the degree of the general form of the bovine jugular vein blood vessel sheet after different method for removing cells are handled, cell free integrated degree, the integrity of collagen elastic fibers preservation, hot shrinkage variation of temperature and maximum strength decline.
1. adopt the PBS liquid of bent that logical (Triton) X-100 of detergent, concentration is less than 1%, and suitable concn is 0.25～0.5%, 37 ℃ and handled the PBS rinsing 24～48 hours down; 2. the PBS of 0.025～0.05% trypsin/0.02%EDTA handles 30～120min down at 37 ℃, the PBS rinsing, and 30～60min is more suitable; 3. the PBS that adds 20u/ml DNase-I and 0.2mg/ml RNase-A handled 24-48 hour down at 37 ℃, and the PBS rinsing places PBS.
3) pipeline places height to ooze PBS liquid (pH:7.6; Soak 2-4h in 600～680mosm), place again to contain 0.01～0.1% methylene blue PBS (pH:7.6; Osm:320mosm) behind middle balance 2～4h, the unsettled irradiation 20～48h of 150～500W electric filament lamp.Reaction system places in 0～25 ℃ of water bath with thermostatic control, and continuous stirring in the irradiation process is blown into air, and the control pH value.Handle fully rinsing decolouring of back.
4) place compound iodine preparation to soak 3 days sterilization treatment or 0.1% bromo geramine, 30～60min, place 50～60% ethanol to preserve again.Compound iodine preparation prescription and processing method are carried out with reference to nineteen ninety-five United States Patent (USP) (patent No. is 5437287).After the 4th step disposed, tissue adopted terylene sterile bag packing, and preserving liquid is DMEM liquid, 10%DMSO and 10%FBS.Organize progressively to be cooled to-80 ℃, under freezing state, (dosage is 25～40kGy), and after the sterilization, tissue low-temperature or room temperature are preserved to adopt the gamma-rays sterilization.
Table 1,
| Grouping | Step 1 | Step 2 | Step 3 |
| I group II group III group IV group V group VI group VII group VIII group XI group X group | The fresh control group, PBS soaks 0.025% trypsin and 0.02%EDTA*, 30min 0.025% trypsin and 0.02%EDTA, 24h 0.25% bent that logical X-100,48h 0.025% trypsin and 0.02%EDTA, 30min 0.25% bent that logical X-100,48h 0.5% bent that logical X-100,48h 0.5% bent that logical X-100,48h 1% bent that logical X-100,48h 1% bent that logical X-100 48h | 20u/mlDNase-I and 0.2mg/ml RNase-A**, 24h 20u/mlDNase-I and 0.2mg/ml RNase-A, 24h 0.025% trypsin and 0.02%EDTA, 30min 0.05% trypsin and 0.02%EDTA, 30min 0.125% trypsin and 0.02%EDTA, 30min 0.05% trypsin and 0.02%EDTA, 30min 0.125% trypsin and 0.02%EDTA 30min | 20u/mlDNase-I and 0.2mg/ml RNase-A**, 24h 20u/mlDNase-I and 0.2mg/ml RNase-A, 24h 20u/mlDNase-I and 0.2mg/ml RNase-A, 24h, 20u/mlDNase-I and 0.2mg/mlRNase-A, 24h 20u/mlDNase-I and 0.2mg/ml RNase-A, 24h |
* contain 0.02%EDTA in 0.025% (0.05% or 0.125%) trypsin solution
20u/mlDNase-I, 0.2mg/ml RNase-A in the * nuclease mixed solution
The result: all valve structures of organizing except III in the bovine jugular vein blood vessel sheet of respectively organizing that take off after the cell processing have the destruction, and the blood vessel wall and the valve structure of other groups are intact, and be similar with fresh group; The hot shrinkage temperature of each group and fresh control group be there was no significant difference relatively.The trypsin 0.125% of excessive concentrations) or the long trypsinization time (24h) can destroy collagen elastic fibers in valve structure and the bovine jugular vein blood vessel, make maximum strength descend more than 20% than fresh control group; That logical X-100 (0.25%) of simple song adds the trypsin 0.025% of low concentration) take off the bovine jugular vein blood vessel sheet residual fraction cellularity that cell is handled; The gentle rapid detergent-enzyme digestion of multistep (0.25% or 0.5% bent that logical X-100,0.025% or 0.05% trypsin and 0.02%EDTA, 20u/mlDNase-I and 0.2mg/ml RNase-A) band lobe bovine jugular vein blood vessel sheet after taking off cell and handling is more satisfactory: blood vessel wall is with the valve general form and handle preceding similar, light microscopic and Electron microscope showed cell component are removed fully, and the collagen elastic fibers is preserved the complete Fig. 3 that sees.
Conclusion: the band lobe bovine jugular vein acellular matrix that the rapid detergent-enzyme digestion of multistep (bent that logical X-100, trypsin and nuclease) takes off after cell is handled is more satisfactory scaffold for vascular tissue engineering.
Embodiment 2
The bovine jugular vein blood vessel tensile strength research of going the cell photooxidation to handle.
Method: gather 20 of local bovine jugular veins, every blood vessel is divided into five sections, is divided into fresh group at random, goes groups of cells, the photooxidation group, goes cell photooxidation group, GA group.Every group of n=20.The glutaraldehyde group adopts 0.625% glutaraldehyde to fix 6 hours, and 0.3% glutaraldehyde is preserved; The photooxidation group adopts 0.1% methylene blue as the mediation agent, and light application time is 48 hours, and intensity of illumination is 4000 lumen-hours, and mediation agent temperature is 12-18 ℃, the compound iodine preparation sterilization, and 60% ethanol is preserved; Go cell to adopt 0.5% bent that logical X-100 to handle 0.05% trypsin and 0.02%EDTA, 30min and 20u/mlDNase-I and 0.2mg/ml RNase-A, 24h 48 hours in conjunction with the photooxidation group.PBS liquid rinsing repeatedly (i.e. VII prescription case in the table 1) between each step.Adopt with the constructed further dyestuff mediation photooxidation of photooxidation group and handle, the compound iodine preparation sterilization, 60% ethanol is preserved.Fresh group of blood vessel adopts 60% ethanol to preserve.Locate walk crosswise cutting long 4cm, the vascular strip of wide 0.5cm with bovine jugular vein more than apart from lobe hole 1cm from all experiments.Adopt the biological tensiometer of STRON to measure the biomechanical property of bovine jugular vein vascular strip.
Result: the maximum stress such as the following table 2 of each group.Go the maximum stress of cell photooxidation processed group significantly to be better than fresh group.Go cell to handle counter stress and do not have obvious influence.But the blood vessel stress that goes the cell photooxidation to handle significantly is lower than the glutaraldehyde cross-linking group.
Table 2, the maximum stress of the bovine jugular vein blood vessel sheet that different technologies is handled are relatively
| Fresh group | Go groups of cells | The photooxidation group | Go cell photooxidation group | The GA group | |
| Maximum stress (N) | 28.2±4.2 | 27.6±3.2 | 45.8±3.8 | 42.6±4.2 | 60.1±7.2 |
Conclusion: go cell to improve the tensile strength of organizing, but the tensile strength of the material that too late glutaraldehyde is handled is big in conjunction with photooxidation Processing of Preparation bovine jugular vein.
Embodiment 3
The bovine jugular vein histone of different technologies Processing of Preparation extracts test
Method: gather local bovine jugular vein, adopt glutaraldehyde cross-linking, photooxidation is crosslinked and remove the crosslinked preparation bovine jugular vein of cell photooxidation (method is with embodiment 2).The bovine jugular vein of different technologies preparation and fresh bovine jugular vein and the bovine jugular vein that goes cell to handle are smashed to pieces, add tissue extract, get 20 μ l extracting solution and make the polyacrylamide gel electrophoresis analysis of protein.
Result: see Fig. 7, compare with fresh pipeline, the pipeline that glutaraldehyde processed group and photooxidation reaction group are handled organizes the protein extraction amount all obviously to reduce, photooxidation reaction is the same with glutaraldehyde that bovine jugular vein is played fixation in this explanation, structure stability significantly increases, and along with the photooxidation reaction increase of action time, the minimizing of carrying out property of histone extracted amount.Go cell to handle structure stability is not had obvious influence.
Embodiment 4
The bovine jugular vein immunogenicity comparative study of different crosslinking technological preparations
Method: gather local bovine jugular vein, adopt glutaraldehyde cross-linking, photooxidation is crosslinked and removes the crosslinked preparation bovine jugular vein of cell photooxidation (concrete scheme is with embodiment 2).The bovine jugular vein of different technologies preparation and fresh bovine jugular vein homogenate respectively extract antigen, are mixed and made into immune new zealand white rabbit behind the multiple emulsion antigen with complete Freund's adjuvant again, and double immunodiffusion is measured the rabbit anteserum antibody titer.Get fresh bovine jugular vein immunity antiserum, the bovine jugular vein valved conduit immunogenicity after adopting immunoblotting (Western blotting) vitro detection being crosslinked.Western blotting method: (1) SDS-PAGE: getting and concentrating gum concentration is 4.0%, and resolving gel concentration is 10% slab-electrophoresis, and every plate adds the bovine jugular vein of fresh bovine jugular vein, PC, PH, GA crosslinking Treatment successively and knits antigen.Tissue antigen before application of sample all with sample buffer with contrast dilution in 1: 1 and heat treated (100 ℃, 4min).After electrophoresis finished, gel was with coomassie brilliant blue staining or carry out electricity commentaries on classics film.(2) electricity changes film: protein band is transferred to nitrocellulose filter (NC film) from gel, and transfer voltage is constant voltage 45V, and be 3h transfer time, and the gel behind the electrotransfer uses coomassie brilliant blue staining to observe the albumen transfer efficiency.(3) immunoblotting reaction: (Tween 80 with the Tween 80/Tris salt buffer that contains 1%BSA, TBS) sealing NC film, then with the NC film successively with 1: 40 serum and 1: the reaction of 12000AP goat anti-rabbit igg, carry out chromogenic reaction with BCIP/NBT as substrate at last, band occur and background when lighter with the distilled water flushing cessation reaction.
Result: antibody dilution to 1: after 1000, the fresh bovine jugular vein organizes antiserum to still have obvious band to show, illustrates that tiring of antibody can reach 1: 1000 at least, can satisfy the needs of experiment fully.The band that fresh bovine jugular vein tissue has specificity to repeat, at least the antigen that comprises 6 different molecular weights, illustrate that fresh bovine jugular vein tissue has antigenicity and antigen to be distributed with specificity, obvious band does not appear substantially through photooxidation and the bovine jugular vein that goes cell to handle in conjunction with photooxidation, 5 groups of bovine jugular veins through the GA crosslinking Treatment have 1 group band to occur, but there is not the band that specificity repeats, illustrate that the bovine jugular vein tissue antigen that photooxidation is handled significantly reduces, through going the antigenicity lower (see figure 8) of cell in conjunction with the bovine jugular vein of photooxidation processing.
Embodiment 5
The bovine jugular vein blood vessel calcification performance study of going the cell photooxidation to handle
Method: gather local bovine jugular vein, adopt glutaraldehyde cross-linking, photooxidation is crosslinked and removes cell photooxidation crosslinking Treatment bovine jugular vein (concrete scheme is with embodiment 2).Be cut into the test piece of 1 * 1.5cm2 band lobe leaf.Selection is from the male SD rat (provided by the Xiangya Medical College, Zhongnan Univ Experimental Animal Center, all meet national laboratory animal regulation) of breast, and subcutaneous embedding was fed 60 days, obtained specimen, and atomic absorption spectrophotometer is measured and organized calcium content.
The result: tube wall and valve calcium content result are respectively 253 ± 37.6,170.6 ± 29.5,121.7 ± 21.8 μ g/mg; 25.6 ± 3.8,21.7 ± 2.9,12.8 ± 2.6 μ g/mg.Relatively between three groups significant difference is arranged all between group.
Conclusion: go the bovine jugular vein of cell combination dye mediation photooxidation Processing of Preparation to significantly improve the calcification performance.
Embodiment 6
The bovine jugular vein pipeline that goes cell to handle in conjunction with photooxidation is rebuild the dog pulmonary artery and is connected with the right ventricle
Method: gather the bovine jugular vein valved conduit, go cell to handle and prepare in conjunction with the photooxidation technology.Use valved conduit and make local domesticated dog outflow tract of right ventricle and rebuild, ligation self pulmonary artery is directly measured pulmonary artery pressure, the right ventricle presses and the body circulation is pressed before and after rebuilding, ultrasound detection in the postoperative seven days, 1 year ultrasonic and cardiac catheter detection hemodynamics of postoperative.
The result: six dogs are rebuild all long-term survivings of back.After rebuilding outflow tract of right ventricle, valve far-end blood pressure is 22/14～37/10mm Hg in the pipeline.Comparing (comprising systolic pressure, diastolic pressure and mean pressure) difference with pulmonary artery pressure before the reconstruction does not have significance (P＞0.05), but diastolic pressure is significantly higher than right ventricular diastolic pressure (p＜0.01) in the pipeline.Rebuilding right ventricle, back blood pressure is 26/5～41/0mm Hg, and relatively preceding with reconstruction, right ventricular diastolic pressure does not have significant change (p＞0.05).But the systolic pressure increase has significance (p＜0.05), and valve is very thin in the postoperative ultrasound detection pipeline, and valve opens and closes good, and many nothings are backflowed or utmost point mild reflux.Pressure gradient is little.Locating extremely after postoperative 1 year and a year and a half, animal obtains the detection of bovine jugular vein specimen, the bovine jugular vein pipeline is unobstructed, and tube wall still has better elasticity, and profile complete (Fig. 4) lobe leaf does not have obviously and thickens, movable good (Fig. 5), tube wall and lobe leaf inner membrance have one deck endothelioid cells growth (Fig. 6).
Conclusion: observe from hemodynamics recent and at a specified future date, self-control bovine jugular vein valved conduit can be used as the good material that outflow tract of right ventricle is rebuild.
Embodiment 7
The plantation of Human umbilical vein endothelial cells on the bovine jugular vein sheet that goes cell to handle in conjunction with photooxidation
Method: gather bovine jugular vein, go cell to handle combination dye mediation photooxidation technical finesse bovine jugular vein.The gamma-rays low temperature sterilization is handled.Obtain Human umbilical vein endothelial cells, the cultivation of going down to posterity is cultivated into endotheliocyte under vascular endothelial cell growth factor etc. is induced.Plantation is cultivated on the bovine jugular vein sheet.
The result: endotheliocyte is planted growth and long-term surviving (Fig. 9) on the bovine jugular vein sheet, and the intensive growth of cell is arranged in order, and is adherent firm.
Conclusion: the bovine jugular vein that goes cell to handle in conjunction with photooxidation is fit to the human endothelial cell growth.
Claims (10)
1, be used for the preparation method of the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction, comprise the steps: 1) obtain bovine jugular vein, and rinsing, sterilization; 2) adopt the photooxidation reaction of dyestuff mediation to make jugular vein substrate crosslinked; 3) sterilization and preservation; It is characterized in that before the 2nd step, spending earlier the cell agent and handle the bovine jugular vein that the 1st step obtained.
2, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 1, the cell processing method of going that it is characterized in that being adopted is to unite the detergent that adopts nonionic and digestive enzyme or/and the method that nuclease is handled; Digestive enzyme is a trypsin; Nuclease is deoxyribonuclease and ribonuclease.
3, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 2 is characterized in that used detergent leads to 100 for bent that.
4, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 3, the concentration that it is characterized in that detergent is 0.25-1%; Concentration is 0.025～0.05% trypsin when digestive enzyme is trypsin; When nuclease was deoxyribonuclease and ribonuclease, its concentration was respectively 10-40unit/ml and 0.1-0.4mg/ml.
5, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 4, it is characterized in that the used detergent-treatment time is 24-48 hour, the trypsin treatment time is 30--120 minute, and deoxyribonuclease and ribonuclease mixing post processing time are 24-72 hour.
6, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 5, it is characterized in that bent those logical 100 concentration are 0.05%, processing time is 48 hours, digestive enzyme is a trypsin, its concentration is 0.025-0.125%, and the processing time is 30--60 minute; And deoxyribonuclease and ribonuclease, its concentration is respectively 20u/ml and 0.2mg/ml, and the processing time is 24 hours.
7, according to the described preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction of one of claim 1 to 6, it is characterized in that adopting methylene blue in the photooxidation reaction, and at pH:7.6; Under the 0sm:320mosm condition, the unsettled irradiation 20～48h of 150～500W electric filament lamp.
8, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 7 is characterized in that in methylene blue solution pH:7.6; Soaked 2-4 hour under the 0sm:320mosm condition.
9, the preparation method that is used for the porcine cardiovascular conduit of pulmonary artery reparation or reconstruction according to claim 7 is characterized in that when carrying out photooxidation reaction, and porcine cardiovascular conduit is in and is not higher than under 25 ℃ of temperature conditions.
10, a kind of porcine cardiovascular conduit that is used for pulmonary artery reparation or reconstruction is characterized in that it is according to the preparation of the method for one of claim 1-9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104095692A (en) * | 2013-04-08 | 2014-10-15 | 天津市塑料研究所有限公司 | Method for manufacturing bovine jugular vein valved conduit |
| CN105169479A (en) * | 2015-07-29 | 2015-12-23 | 中南大学湘雅二医院 | Preparation method of externally constructed tissue engineering trachea matrix |
| CN105833356A (en) * | 2016-05-09 | 2016-08-10 | 拜欧迪赛尔（北京）生物科技有限公司 | Novel acellular valved vascular stent and preparation method thereof |
| CN114504682A (en) * | 2021-11-16 | 2022-05-17 | 李平 | Application of umbilical artery in preparation of coronary bypass graft blood vessel material and method for preparing coronary bypass graft blood vessel material |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6214054B1 (en) * | 1998-09-21 | 2001-04-10 | Edwards Lifesciences Corporation | Method for fixation of biological tissues having mitigated propensity for post-implantation calcification and thrombosis and bioprosthetic devices prepared thereby |
| US6723335B1 (en) * | 2000-04-07 | 2004-04-20 | Jeffrey William Moehlenbruck | Methods and compositions for treating intervertebral disc degeneration |
| CN1439431A (en) * | 2003-04-01 | 2003-09-03 | 北京市普惠生物医学工程公司 | Photo-oxidation of collagen tissue of heterogenic implant of heart |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104095692A (en) * | 2013-04-08 | 2014-10-15 | 天津市塑料研究所有限公司 | Method for manufacturing bovine jugular vein valved conduit |
| CN105169479A (en) * | 2015-07-29 | 2015-12-23 | 中南大学湘雅二医院 | Preparation method of externally constructed tissue engineering trachea matrix |
| CN105833356A (en) * | 2016-05-09 | 2016-08-10 | 拜欧迪赛尔（北京）生物科技有限公司 | Novel acellular valved vascular stent and preparation method thereof |
| CN105833356B (en) * | 2016-05-09 | 2018-02-16 | 拜欧迪赛尔（北京）生物科技有限公司 | A kind of new de- cell Valved allograft support and preparation method thereof |
| CN114504682A (en) * | 2021-11-16 | 2022-05-17 | 李平 | Application of umbilical artery in preparation of coronary bypass graft blood vessel material and method for preparing coronary bypass graft blood vessel material |
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| CN100443064C (en) | 2008-12-17 |
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