CN100586490C - Biological tissue material for artificial esophagus and preparation method thereof - Google Patents
Biological tissue material for artificial esophagus and preparation method thereof Download PDFInfo
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- CN100586490C CN100586490C CN200710049251A CN200710049251A CN100586490C CN 100586490 C CN100586490 C CN 100586490C CN 200710049251 A CN200710049251 A CN 200710049251A CN 200710049251 A CN200710049251 A CN 200710049251A CN 100586490 C CN100586490 C CN 100586490C
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Abstract
本发明公开的生物组织材料是一种以猪主动脉血管为基体，以京尼平为交联剂制备的人工食道用生物组织材料，其制备的主要工序为：(1)将自屠宰场活杀猪体上取下的主动脉血管进行清洗，去除血迹和脂肪；(2)将清洗干净的猪主动脉血管用由缓冲液和京尼平交联剂配制成的交联液进行交联处理，交联体系的京尼平浓度为2.70～4.50mmol/100ml，pH值为3.5-7.4，温度为20-28℃。经充分交联制备的人工食道用生物组织材料，既具有高分子材料富有韧性、弹性好的力学特征，又有适于周围细胞长入的天然结构，还有类似生物活性蛋白的促使细胞在其表面黏附生长的特点，预期应用于临床可大大降低食管切除替换术后吻合口漏、狭窄及坏死等并发症。The biological tissue material disclosed by the present invention is a biological tissue material for artificial esophagus prepared with pig aortic blood vessels as the matrix and genipin as the cross-linking agent. The aortic blood vessel removed from the slaughtered pig body is cleaned to remove blood and fat; (2) the cleaned pig aortic blood vessel is cross-linked with a cross-linking solution prepared by buffer and genipin cross-linking agent , the concentration of genipin in the cross-linking system is 2.70-4.50mmol/100ml, the pH value is 3.5-7.4, and the temperature is 20-28°C. The biological tissue material for artificial esophagus prepared by fully cross-linking not only has the mechanical characteristics of toughness and good elasticity of polymer materials, but also has a natural structure suitable for the growth of surrounding cells, and it also has the ability to promote cells in its The characteristics of surface adhesion growth are expected to greatly reduce complications such as anastomotic leakage, stenosis and necrosis after esophagectomy and replacement.
Description
Technical field
The present invention relates to human organ and substitute artificial bio-membrane's organization material technology, more particularly, relate to a kind of biological tissue material for artificial esophagus technology, belong to technical field of biological material.
Technical background
Some disease of esophagus aspect as the treatment of the esophageal carcinoma, esophagus benign stricture and congenital esophageal atresia etc., all is to adopt operative treatment usually.The traditional method of operative treatment is the esophagus that substitutes excision with other histoorgans of patient self.This replacement therapy method is big to patient trauma, and postoperative can bring such as a series of complication such as anastomotic leakage, narrow and necrosis, simultaneously because the sacrificial section gastrointestinal to substitute esophagus, causes patient that digestive functional disturbance and nutrient malabsorption usually take place.People are seeking a kind of ideal substitute (artificial esophagus) always and are overcoming above-mentioned shortcoming for this reason.
The beginning of artificial esophagus research has been opened up in the experiment of nineteen fifty-two Berman, since half a century, the countries in the world scholar has carried out extensive studies to the artificial material esophagus, obtained numerous achievements in research, but still there are problems in the achievement in research of prior art, still do not have ideal artificial esophagus so far and come out, research still is in animal experiment stage basically.The starting stage of artificial material esophagus research, people adopt the macromolecular material artificial esophagus to substitute the esophagus of excision.The subject matter that the macromolecular material esophagus exists is anastomotic leakage, narrow and necrosis etc., and these problems can not be well solved.Subsequently, have the scholar with the compound artificial esophagus of making of macromolecular material and biological tissue's (as collagen) promoting its biocompatibility, but effect is still undesirable.In the research of artificial material esophagus, some natural biological tissue such as small intestinal submucosa also are used to the research of this area, but narrow complication still often takes place.The engineered artificial esophagus that latest developments are got up overcome anastomotic leakage, there is good performance aspect such as narrow, but its body plan process complexity, the specification requirement height is difficult to realize industrialization.In general, in order to solve above-mentioned a series of complication, the new substitution material of exploitation of still needing.
Summary of the invention
The deficiency that exists at the biological tissue material for artificial esophagus of prior art, purpose of the present invention aims to provide that a kind of both to have had macromolecular material artificial esophagus mechanical property good, have again to be suitable for the natural structure that peripheral cell grows into and to impel cell, to overcome or to reduce the esophagus excision and replace complication problems such as anastomotic leakage after the operation, narrow and necrosis at biological tissue material for artificial esophagus of the similar biological activity protein of its surface adhesion growth and preparation method thereof.
Can realize the porcine aorta blood vessel of biological tissue material for artificial esophagus for crossing of foregoing invention purpose, in particular for the pig thoracic aorta blood vessel of crossing with the biological cross-linking agent crosslinking Treatment of genipin with the biological cross-linking agent crosslinking Treatment of genipin.
As the porcine aorta blood vessel of crossing with the biological cross-linking agent crosslinking Treatment of genipin of biological tissue material for artificial esophagus, its preparation processing method mainly comprises following manufacturing procedure:
(1) work that the suicide pig farm is obtained is killed the porcine aorta blood vessel and is cleaned removal bloodstain and fat;
(2) it is that the genipin of the buffer of 3.5-7.4 and adding is mixed with crosslinked fluid and carries out crosslinking Treatment that the porcine aorta blood vessel that cleans up is used by pH value, the pH value of cross-linking system is 3.5-7.4, crosslinking temperature is 20-28 ℃, the concentration of genipin is 2.70～4.50mmol/100ml in the crosslinked fluid, and the consumption of crosslinked fluid is every 1cm
2Tissue 5ml-10ml, through the full cross-linked biological tissue material for artificial esophagus for preparing.
In above-mentioned preparation processing scheme, taking from the bloodstain on the aorta vessel of pig live body and the cleaning of fat removes, preferably do not ooze out to there being blood and slime with normal saline flushing earlier, the back is the benzalkonium bromide aqueous solution sterilization degrease of 0.01%-0.05% with mass concentration, and benzalkonium bromide is removed in reuse phosphate buffer flushing afterwards.The benzalkonium bromide aqueous solution can add clear water by commercially available benzalkonium bromide solution (mass concentration 5%) and be mixed with.
In above-mentioned preparation processing scheme, the buffer of preparation genipin crosslinked fluid is the citric acid/sodium citrate buffer.The citric acid/sodium citrate buffer can be by 1: 1.8～2.0 preparations of pressing ratio of quality and the number of copies of the sodium citrate solution of the citric acid solution of 0.1M and 0.1M.
In above-mentioned preparation processing scheme, the crosslinked of aorta vessel and genipin crosslinked fluid preferably carries out crosslinking Treatment having under the condition of slight vibration, so that it is more abundant and even that crosslinked fluid can contact with the tissue that is crosslinked, normally in shaking table, carry out crosslinking Treatment.
In above-mentioned preparation processing scheme, the porcine aorta blood vessel is fetched into the time of carrying out crosslinking Treatment and generally is controlled to be and is not more than 6h from the pig live body.
After the porcine aorta blood vessel is prepared as biological tissue material for artificial esophagus with the genipin crosslinked fluid is crosslinked, need be stored in the alcoholic solution of 30%-50% (volume ratio of dehydrated alcohol and water is 1: 2～1: 1) standby.Material also must carry out cobalt 60 illumination-based disinfections when using or carrying out experiment made on the living or cell in vitro related experiment.
In the research process of preparation biological tissue material for artificial esophagus, the inventor is through repeated tests with to the profound understanding of cross-linking agent performance, and selected genipin cross-linking agent is as the cross-linking agent of porcine aorta blood vessel.The genipin cross-linking agent is a kind of heterocyclic compound that obtains that extracted by pure-natural biological, (COO-) etc. a plurality of active functional group group that has (OH), can spontaneously react with aminoacid or protein, generate an iridoid nitride, form an aromatic monomer through a dehydration subsequently, this monomer is cyclic intermolecular and intramolecular crosslinking structure owing to its radical reaction forms.Crosslinked mechanism is seen accompanying drawing 1.Genipin has advantages such as cytotoxicity is low, little to natural tissues destruction, cellular affinity is strong as a kind of natural biological cross-linking agent.The biological organization material of handling with genipin, antigenicity is removed more thorough, and biological stability is higher, and external antienzyme degradation capability is better, denaturation temperature is lower than material that glutaraldehyde handled and the material of obviously handling greater than epoxy polymer, and ultimate tensile strength is better.Compare with other chemical cross-linking agents, the great advantage of genipin is a low cytotoxicity.Cross-linking agent cytotoxicity by the MTT test determination shows that the genipin cytotoxicity is littler 10000 times than glutaraldehyde, also is significantly less than epoxy polymer.
The present invention is a cross-linking agent with biological cross-linking agent genipin, with the porcine aorta is the prepared biological tissue material for artificial esophagus of matrix, both had macromolecular material and be rich in the mechanical characteristics of toughness, good springiness, have again and be suitable for the natural structure that peripheral cell is grown into, the characteristics of impelling cell to grow that also have similar biological activity protein at its surface adhesion, can reduce esophagus excision greatly and replace complication such as anastomotic fistula, narrow and necrosis, be well suited for using material as the artificial esophagus.And its preparation process is simple, and specification requirement is not high, easily realizes industrialization.
Description of drawings
Accompanying drawing 1 is the crosslinked mechanism sketch map of genipin.
Accompanying drawing 2 goes cell situation (HE dyeing is amplified 100 times) for crosslinked front and back material.2-1 fresh sample wherein; 2-2 is for being 7.4 with genipin at PH, and crosslinked fluid concentration is crosslinked 7 days situation under the condition of 2.76mmol%; 2-3 is for being 4.0 with genipin at PH, and crosslinked fluid concentration is crosslinked 7 days situation under the condition of 2.76mmol%.
Accompanying drawing 3 is the situation of change (the Mallory trichrome stain amplifies 100 times) of the collagen fiber of crosslinked front and back material.The 3-1 fresh sample; 3-2 is for being 7.4 with genipin at PH, and crosslinked fluid concentration is crosslinked 7 days situation under the condition of 2.76mmol%; 3-3 is for being 4.0 with genipin at PH, and crosslinked fluid concentration is crosslinked 7 days situation under the condition of 2.76mmol%.
Accompanying drawing 4 is the situation of change (Verhoeff Garapa uniformly dyeing color method amplifies 100 times) of the elastic fiber of crosslinked front and back material.The 4-1 fresh sample; 4-2 is for being 7.4 with genipin at PH, and crosslinked fluid concentration is crosslinked 7 days situation under the condition of 2.76mmol%; 4-3 is for being 4.0 with genipin at PH, and crosslinked fluid concentration is crosslinked 7 days situation under the condition of 2.76mmol%.
The specific embodiment
1, artificial esophagus prepares embodiment with biological organization material
Describe the specific embodiment of the present invention by the following examples, content of the present invention is described in further detail again.But of particular note, this should be interpreted as protection scope of the present invention only limits to embodiment, be on the contrary, allly should all belong to protection scope of the present invention based on the technology that content of the present invention realized.
In following each embodiment, except that specifying, related percentage ratio is mass percent, and related umber is mass fraction.
Embodiment 1
The suicide pig farm is taken out to live and is killed the thoracic aorta blood vessel of pig, puts into brine ice and preserves, and transfers to laboratory rapidly.Remove the fatty tissue and the reticular connective tissue of surface attachment.Wash repeatedly with a large amount of normal saline earlier, clean bloodstain and fat, till no blood and slime oozes out.Be about 0.03% benzalkonium bromide aqueous solution sterilization degrease about 45 minutes with mass concentration afterwards, the reuse phosphate buffer washes repeatedly removes clean benzalkonium bromide.The benzalkonium bromide aqueous solution is that 5% benzalkonium bromide solution adds clear water and is mixed with by commercially available mass concentration.
Porcine aorta blood vessel of rinsing well and crosslinked fluid place under the condition of vibration gently, carry out in the shaking table crosslinked so that crosslinked fluid be crosslinked organize abundant more with contact equably.Take out aorta vessel to carrying out crosslinking Treatment from the pig live body, the time control between this is no more than 6 hours.Crosslinked condition is: crosslinking temperature is a room temperature, and promptly temperature is about 20-28 ℃; The pH value of cross-linking system is about 4.0; Crosslinked fluid is that about 4.0 buffer adds the genipin cross-linking agent and is mixed with by pH value, and concentration is the genipin that contains in the 100ml crosslinked fluid about 2.76 μ mol; Crosslinking time is 7 days.Buffer in crosslinked fluid preparation: prepare the citric acid solution of 0.1M and the sodium citrate solution of 0.1M respectively, get 1 part of sodium citrate solution and add about 1.9 parts citric acid solution again and be mixed with the citric acid/sodium citrate buffer.The consumption of crosslinked fluid is that the tissue of every 1cm2 is handled with about 5ml-10ml crosslinked fluid.
With the porcine aorta blood vessel is that matrix is the artificial esophagus biological organization material of cross-linking agent with the genipin, is stored in volume ratio after crosslinked being prepared into and is in the alcoholic solution about 30% (volume ratio of dehydrated alcohol and water is 1: 2) standby.When cross-linked material will carry out experiment made on the living or cell in vitro related experiment, must carry out cobalt 60 illumination-based disinfections.
The crosslinked cross-linking agent that both can save costliness can reach ideal cross-linking effect again under this condition.
Embodiment 2
What present embodiment prepared is matrix with the porcine aorta blood vessel, with the genipin is the biological tissue material for artificial esophagus of cross-linking agent, its preparation process is substantially the same manner as Example 1, different porcine aorta blood vessel kill the descending aorta blood vessel of pig for living, the mass concentration of degreasant benzalkonium bromide aqueous solution of being used to sterilize is about 0.05%, the pH value of cross-linking system is about 4.0, the pH value of crosslinked fluid is about 4.0, and the genipin concentration in the crosslinked fluid contains genipin about 4.42 μ mol for the 100ml crosslinked fluid; Crosslinking time is 8 days.The preparation of buffer: the first citric acid solution of preparation 0.1M, prepare the sodium citrate solution of 0.1M then, get 1 part of sodium citrate solution and add about 2.1 parts of citric acid solutions again and be mixed with the citric acid/sodium citrate buffer.The consumption of crosslinked fluid is every 1cm
2Tissue handle with about 5ml-10ml crosslinked fluid.
It is standby in the alcoholic solution about 50% (volume ratio of dehydrated alcohol and water is 1: 1) that the crosslinked cross-linked material that is prepared into is stored in volume ratio.When carrying out experiment made on the living or cell in vitro related experiment, cross-linked material also must carry out cobalt 60 illumination-based disinfections.
Embodiment 3
What present embodiment prepared is matrix with the porcine aorta blood vessel, with the genipin is the biological tissue material for artificial esophagus of cross-linking agent, its preparation process is substantially the same manner as Example 1, different porcine aorta blood vessel kill the descending aorta blood vessel of pig for living, the mass concentration of degreasant benzalkonium bromide aqueous solution of being used to sterilize is about 0.04%, the pH value of cross-linking system is about 7.4, the pH value of crosslinked fluid is about 7.4, and the genipin concentration in the crosslinked fluid contains genipin about 2.76 μ mol for the 100ml crosslinked fluid; Crosslinking time is 6 days.The preparation of buffer: the NaCl of preparation 0.14M in a distilled water, the KCl of 0.003M, the Na of 0.01M
2HPO
4And the KH of 0.0016M
2PO
4, reuse HCl regulates PH to 7.4.The consumption of crosslinked fluid is every 1cm
2Tissue handle with the 5ml-10ml crosslinked fluid.
It is standby in the alcoholic solution about 50% (volume ratio of dehydrated alcohol and water is 1: 1) that the crosslinked cross-linked material that is prepared into is stored in volume ratio.When carrying out experiment made on the living or cell in vitro related experiment, cross-linked material also must carry out cobalt
60Illumination-based disinfection.
2, artificial esophagus biological organization material testing performance index embodiment
(1) degree of cross linking test implementation example
The degree of cross linking is an important indicator of reflection crosslinking degree, and it is represented by the ratio of the free amino group content in the free amino group content in the crosslinked back tissue and the uncrosslinked tissue.Because the crosslinked of genipin will consume free amino groups a large amount of in the tissue, so the degree of cross linking is big more, free amino group residual in the tissue is just few more.Above-mentioned artificial esophagus is measured with the degree of cross linking that biological organization material prepares the cross-linked material of embodiment preparation, adopted indenes three bronze medal methods to carry out.
The degree of cross linking of cross-linked material under the table 1-different condition
As can be seen from the above table, in the time that communicates,, under the concentration 2.76 μ mol% conditions, crosslinked the most thorough at the soonest at pH4.0.Three kinds of crosslinked conditions all can reach the higher degree of cross linking after 7 days.The high more illustrative material of the degree of cross linking is crosslinked good more, and the free amino group in the biological tissue is removed thorough more, and the antigenicity of biological tissue is removed also thorough more, and it is weak more that material is implanted the immunological rejection that produces behind the live body
(2) denaturation temperature test implementation example
Another important indicator of the crosslinked situation of biological organization material is a denaturation temperature.Above-mentioned artificial esophagus is measured with the denaturation temperature that biological organization material prepares the cross-linked material of embodiment preparation, adopted differential scanning calorimeter to measure.
The denaturation temperature of cross-linked material under the table 2-different condition
From last table as seen, crosslinked its denaturation temperature of biomaterial of genipin is all higher, and this illustrates the Heat stability is good of this kind material, and the crosslinked crosslinked more thorough than epoxy polymer of genipin also is described.
(3) Mechanics Performance Testing embodiment
As the bioengineered tissue material that the artificial esophagus uses, the mechanical property of tissue is a crucial index.Above-mentioned artificial esophagus is measured with the mechanical property that biological organization material prepares the cross-linked material of embodiment preparation, adopted the 302instron universal material experimental machine of INSTRON company to measure.
The mechanical property of cross-linked material under the table 3-different condition
We can see from top data, more crosslinked mechanical properties that can improve material, and modulus wherein, maximum tensional stress all is significantly improved, and this is wherein with at PH4.0, the sample increase rate maximum of handling under the condition of crosslinked fluid concentration 2.76mmol%.And, reduce to some extent in the strain at maximum stress place.Tissue after this explanation is crosslinked shows slightly stiff, and the elasticity of fresh tissue is better, and soft.
The document record shows, the mechanical property of human esophagus and intensity, and maximum tensional stress is approximately 1.3 ± 0.5MPa.From the data that last table Mechanics Performance Testing obtains, we can see, the mechanical property of the tissue after crosslinked can satisfy artificial esophagus's mechanical property requirements substantially.
(4) cytotoxicity test implementation example:
Reach ideal clinical effectiveness, also must make material that good cell compatibility is arranged.Above-mentioned artificial esophagus being prepared the Cytotoxic measurement of cross-linked material that embodiment prepares with biological organization material, adopt the tetrazolium salts colorimetry, serves as to detect cell with the L-929 cell, and crosslinked material is carried out cell toxicity test.The result is as follows:
The cytotoxicity of cross-linked material under the table 4-different condition
Annotate: condition 1 is pH4.0, concentration 2.76mmol%, and condition 2 is pH7.4, concentration 2.76mmol%.
Cytotoxicity is classified as, and 0～1 grade is qualified, so the cell compatibility of this cross-linked material is qualified.
3, artificial esophagus biological organization material Micro-Structure Analysis embodiment
Go cell situation, collagen fiber situation of change, elastic fiber situation of change etc. also very important after biological organization material is crosslinked to cross-linked material.The variation of these situations can obtain by the light microscopic photo relative analysis to the crosslinked front and back of bioengineered tissue material.
(1) crosslinked front and back material removes cell situation Micro-Structure Analysis embodiment
The crosslinked front and back of porcine aorta blood vessel material goes the cell situation to see the light microscopic photo shown in accompanying drawing 2-1,2-2, the 2-3.Can find out that from the light microscopic photo structure of flesh tissue is very complete, have much to have the nuclear cell of purple.At condition PH is 7.4, and crosslinked fluid concentration is in the crosslinked fluid of 2.76mmol%, and crosslinked seven days sample has been removed a part of cell.And be 4.0 at condition PH, crosslinked fluid concentration is in the crosslinked fluid of 2.76mmol%, and crosslinked seven days sample, the cell component of the overwhelming majority have been removed, and the removal effect of pair cell is more obvious.Cell is that tissue produces one of antigenic main source, and its removal is thorough more, and the immunogenicity that crosslinked tissue produces is just more little.
(2) the collagen fiber situation of change Micro-Structure Analysis embodiment of crosslinked front and back material
The collagen fiber situation of change of the crosslinked front and back of porcine aorta blood vessel material is seen the light microscopic photo shown in accompanying drawing 3-1,3-2, the 3-3.Section adopts the Mallory trichrome staining to make.Can find out that from the light microscopic photo collagen fiber of fresh sample are very complete.And be 4.0 and PH7.4 at condition PH, concentration is the crosslinked cross-linked material that obtained in seven days in the crosslinked fluid of 2.76mmol%, it is all very complete that collagen fiber are preserved, and is not damaged.In addition, also can find out at PH to be crosslinked under 4.0 the condition, its effect of removing cell is stronger.
(3) the elastic fiber situation of change Micro-Structure Analysis embodiment of crosslinked front and back material
The elastic fiber situation of change of the crosslinked front and back of porcine aorta blood vessel material is seen the light microscopic photo shown in accompanying drawing 4-1,4-2, the 4-3.Section adopts Verhoeff Garapa uniformly dyeing color method to make.Can find out that from the light microscopic photo elastic fibers of fresh sample is very complete.And be 4.0 and 7.4 at condition PH, crosslinked three days sample in the crosslinked fluid of concentration 2.76mmol%, it is all very complete that elastic fibers is preserved.Slightly different is, the sample elastic fibers after crosslinked has become stiff a bit a little, and the tissue after therefore crosslinked shows slightly stiff.And to be that the elastic fibers of sample crosslinked under 4.0 conditions becomes stiff degree smaller for PH by contrast, and degree of crook and flesh tissue are close.
For by the biological organization material handled, we wish under the situation of not destroying collagen fiber and elastic fibers microstructure strongly, and some can produce immunoreactive bioactive substance to remove cell etc.Can find out that from the situation of top several figure the situation in cell place to go is better in the tissue after crosslinked, and microstructure preserves better, collagen fiber and elastic fibers all do not have destroyed.
Claims (9)
1, a kind of biological tissue material for artificial esophagus is characterized in that biological organization material is the porcine aorta blood vessel of crossing through the biological cross-linking agent crosslinking Treatment of genipin.
2, biological tissue material for artificial esophagus according to claim 1 is characterized in that said porcine aorta blood vessel is a pig descending aorta blood vessel.
3, for the processing method of claim 1 or 2 described biological tissue material for artificial esophagus, it is characterized in that comprising following operation:
(1) work that the suicide pig farm is obtained is killed the porcine aorta blood vessel and is cleaned removal bloodstain and fat;
(2) it is that the crosslinked fluid that the genipin of the buffer of 3.5-7.4 and adding is mixed with carries out crosslinking Treatment that the porcine aorta blood vessel that cleans up is used by pH value, the pH value of cross-linking system is 3.5-7.4, crosslinking temperature is 20-28 ℃, the concentration of genipin is 2.70～4.50mmol/100ml in the crosslinked fluid, and the consumption of crosslinked fluid is every 1cm
2Tissue 5ml-10ml, through the full cross-linked biological tissue material for artificial esophagus that promptly is prepared into.
4, the processing method of biological tissue material for artificial esophagus according to claim 3, it is characterized in that bloodstain and fatty cleaning removal on the described aorta vessel, be not ooze out to there being blood and slime with normal saline flushing earlier, the back is the benzalkonium bromide aqueous solution sterilization degrease of 0.01%-0.05% with mass concentration, and benzalkonium bromide is removed in reuse phosphate buffer flushing afterwards.
5, the processing method of biological tissue material for artificial esophagus according to claim 3, the buffer that it is characterized in that preparing the genipin crosslinked fluid is the citric acid/sodium citrate buffer.
6, the processing method of biological tissue material for artificial esophagus according to claim 5 is characterized in that described citric acid/sodium citrate buffer is formulated by ratio of quality and the number of copies 1: 1.8～2.0 by the sodium citrate solution of the citric acid solution of 0.1M and 0.1M.
7, the processing method of biological tissue material for artificial esophagus according to claim 3 is characterized in that aorta vessel and genipin crosslinked fluid are to carry out crosslinking Treatment under the condition of vibration.
8, the processing method of biological tissue material for artificial esophagus according to claim 7 is characterized in that aorta vessel and genipin crosslinked fluid carry out crosslinking Treatment in shaking table.
9, the processing method of biological tissue material for artificial esophagus according to claim 3 is characterized in that the porcine aorta blood vessel is fetched into the time of carrying out crosslinking Treatment and is not more than 6h from the pig live body.
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| CN105056306B (en) * | 2013-04-17 | 2020-04-28 | 拓睿美（北京）医疗科技有限公司 | Application of material prepared by genipin cross-linking intestinal submucosa |
| CN107014658B (en) * | 2017-05-27 | 2023-08-25 | 四川省肿瘤医院 | Method and kit for extracting free cells |
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| CN101766842B (en) * | 2010-03-22 | 2013-11-06 | 四川大学 | Biological tissue material for artificial organs and preparation method thereof |
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