CN103230309A - Tissue engineering vessel and preparation method and application thereof - Google Patents
Tissue engineering vessel and preparation method and application thereof Download PDFInfo
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- CN103230309A CN103230309A CN2013101634777A CN201310163477A CN103230309A CN 103230309 A CN103230309 A CN 103230309A CN 2013101634777 A CN2013101634777 A CN 2013101634777A CN 201310163477 A CN201310163477 A CN 201310163477A CN 103230309 A CN103230309 A CN 103230309A
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Abstract
The invention relates to a tissue engineering vessel and a preparation method and an application thereof. The tissue engineering vessel uses a tubular gelatin/polycaprolactone electricity texture membrane as a support, and smooth muscle cells are evenly distributed in the support. The preparation method includes: a) spreading disinfected gelatin/polycaprolactone materials in a culture dish; b) collecting umbilical artery smooth muscle cells, evenly inoculating the umbilical artery smooth muscle cells on the surface of the gelatin/polycaprolactone electricity texture membrane to culture over the night, and forming a cell material composite electricity texture membrane; and c) twisting the cell material composite electricity texture membrane on a polyvinyl chloride (PVC) tube for culturing, and finally forming the tubular tissue engineering vessel. Cells of the tissue engineering vessel prepared by the method are evenly distributed, simultaneously the tissue engineering vessel has the advantages of being good in elasticity, moderate in hardness and compact in tissue structure, having smooth inner walls and abundant collagenous fibers, and being clear in self tissue boundary and the like. The characteristics of the tissue engineering vessel are close to that of living body vessels.
Description
Technical field
The present invention relates to the tissue engineering technique field, specifically, relate to a kind of engineering blood vessel and its production and use.
Background technology
Angiopathy is one of the highest disease of the present mortality rate of China, and blood vessel transplantation is its main surgical intervention means.Need to utilize engineering blood vessel to substitute the sufferer blood vessel in the vascular transplant.Engineering blood vessel is to utilize the tissue engineering method, and is compound with blood vessel seed cell (endotheliocyte, smooth muscle cell) and relevant cell epimatrix (timbering material), to make up from the form to the function all the engineered blood vessel near intravital blood vessel.Advantages such as that the engineering blood vessel of function admirable should possess is clear with autologous tissue boundary, have certain elasticity and hardness, organizational structure densification, cell distribution are even are similar to the function of intravital blood vessel with performance.
At present, gelatin and polycaprolactone are mixed electrospinning become nano fibrous membrane to be widely used in engineered different tissue, as skin, nerve, bone, blood vessel etc.People such as Chong [Chong EJ, et al. Acta Biomaterialia 2007,3 (3): 321-330] with the research of gelatin/polycaprolactone blending nano fibrous membrane for skin wound healing, the result shows that prepared gelatin/polycaprolactone fibrous membrane obviously is conducive to adhesion, increment and the differentiation of fibroblast.People [Laleh Ghasemi-Mobarakeh such as Laleh Ghasemi-Mobarakeh, et al. Biomaterials 2008,29 (34): 4532-4539] gelatin/polycaprolactone electrospinning is become the very high nano fibrous membrane of the depth of parallelism be used for the reparation of nervous tissue, the cultivation results of neural stem cell shows, gelatin/polycaprolactone nano fibrous membrane is a kind of good tissue engineering scaffold material, the parallel nanofiber that spins is conducive to increment and the differentiation of neurocyte, and is conducive to the elongation of nerve synapse.People [Sepideh Heydarkhan-Hagvall such as Sepideh Heydarkhan-Hagvall, et al. Biomaterials 2008,29 (19): 2907-2914] gelatin is become three-dimensional tissue's engineering rack also successfully be used for the cardiovascular organization engineering with the polycaprolactone electrospinning, the result shows that the nano fibrous membrane of blending shows superior mechanical property and the exclusive cellular affinity of natural material that synthetic material has.Its result shows that all gelatin/polycaprolactone is a kind of good timbering material.
But those skilled in the art know that all the thinking that organizational project makes up normally makes up the support carrier with particular organization's shape approximation earlier, and repopulating cell is cultivated then.When for example utilizing gelatin/polycaprolactone as the timbering material tissue engineering vessel at present, usually all be that gelatin mixes the tubular structure that electrospinning becomes to be similar to blood vessel with polycaprolactone, then cell directly is planted in the outer surface of support, and further cultivates.Be the simulation intravital blood vessel, directly electrospinning becomes wall thickness and the intravital blood vessel thickness basically identical of the timbering material of tubulose, totally thicker, be unsuitable for cell migration and propagation, again because cell is directly to be planted in the timbering material outer surface, thereby the inside and outside cell distribution of the engineering blood vessel that causes structure is inhomogeneous, and performance is not good.How to make up cell distribution evenly, all excellent engineering blood vessels are very important for elasticity and every performances such as hardness is suitable,, organizational structure densification clear with the autologous tissue boundary.
Summary of the invention
The objective of the invention is provides a kind of engineering blood vessel at deficiency of the prior art.
One purpose more of the present invention is that the preparation method of above-mentioned engineering blood vessel is provided.
Another purpose of the present invention is that the purposes of above-mentioned engineering blood vessel is provided.
For achieving the above object, the technical scheme taked of the present invention is:
A kind of engineering blood vessel is support with tubulose gelatin/polycaprolactone electrospinning film, and smooth muscle cell is uniformly distributed in the inside of described support.
Described engineering blood vessel prepares in accordance with the following methods:
A) gelatin/polycaprolactone material that disinfects is tiled in the vessel;
B) collect the umbilical artery smooth muscle cell and be inoculated in overnight incubation behind gelatin/polycaprolactone electrospinning film surface uniformly, form cell material composite electrospun film;
C) cell material composite electrospun film is wrapped on the pvc pipe cultivates, finally form the engineering blood vessel of tubulose.
Preferably, the inoculative proportion of umbilical artery smooth muscle cell and gelatin/polycaprolactone electrospinning film is 10 * 10 in the step b)
6Individual cell/(the electrospinning film of 2cm * 12cm); The number of turns that described cell material composite electrospun film is wrapped on the pvc pipe is the 6-7 circle, cultivates for 1 week then to form for the engineering blood vessel of transplanting, and is preferably in this incubation and always changes liquid once every 2 days half amounts.
For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:
The preparation method of aforesaid engineering blood vessel may further comprise the steps:
A) gelatin/polycaprolactone material that disinfects is tiled in the vessel;
B) collect the umbilical artery smooth muscle cell and be inoculated in overnight incubation behind gelatin/polycaprolactone electrospinning film surface uniformly, form cell material composite electrospun film;
C) cell material composite electrospun film is wrapped on the pvc pipe cultivates, finally form the engineering blood vessel of tubulose.
Preferably, the inoculative proportion of umbilical artery smooth muscle cell and gelatin/polycaprolactone electrospinning film is 10 * 10 in the step b)
6Individual cell/(the electrospinning film of 2cm * 12cm); The number of turns that described cell material composite electrospun film is wrapped on the pvc pipe is the 6-7 circle, cultivates for 1 week then to form for the engineering blood vessel of transplanting, and is preferably in this incubation and changes liquid once every 2 days half amounts.
For realizing above-mentioned the 3rd purpose, the technical scheme that the present invention takes is: the purposes of aforesaid engineering blood vessel in the preparation artificial blood vessel.
The invention has the advantages that:
1, the present invention adopts " egg roll " method, gelatin/polycaprolactone electrospinning film of having inoculated smooth muscle cell is wound on the pvc pipe cultivates that obtain can be for the engineering blood vessel of transplanting, reversed the inertial thinking during engineering blood vessel makes up, in prior art, prepare the way that tubular bracket inoculates cell earlier, can guarantee that the engineering blood vessel cell distribution for preparing is more even, in addition, through a series of mensuration, the engineering blood vessel good springiness that shows this method preparation, hardness is moderate, inner wall smooth, the organizational structure densification, collagen fiber are abundant, clear with autologous tissue boundary, namely possess the various good characteristics of engineering blood vessel, its feature is similar to intravital blood vessel more;
2, utilize method tissue engineering vessel of the present invention, can unify to make cell material composite electrospun film earlier, the pvc pipe of choosing different tube diameters then twines, can obtain the uniform engineering blood vessel of different-diameter and cell distribution, satisfying personalized demand, especially particularly suitable for making up the less engineering blood vessel of diameter;
3, method of the present invention provides suitable cell and inoculative proportion, the cell material composite electrospun film of gelatin/polycaprolactone electrospinning film to be wrapped in the number of turns on the pvc pipe, being wrapped in that cell material composite electrospun film on the pvc pipe cultivates into can be for the time of the engineering blood vessel of transplanting, under this condition, can guarantee that the engineering blood vessel that obtains possesses good characteristic.
Description of drawings
Accompanying drawing 1 is " egg roll " method tissue engineering vessel sketch map.
Accompanying drawing 3 is gelatin/polycaprolactone material qualification results.A: gelatin/polycaprolactone electrospinning film is seen substantially; B: human umbilical artery smooth muscle cell; C: simple gelatin/polycaprolactone scanning electron microscope; D: the gelatin/polycaprolactone scanning electron microscope behind the inoculation smooth muscle cell.
Accompanying drawing 4 is engineering blood vessel cylinder mature and qualification result.A: the sight substantially in 8 weeks in the engineering blood vessel nude mouse (band PVC catheter); B, C: the sight substantially (taking down the PVC catheter) in 8 weeks in the engineering blood vessel nude mouse; D:HE dyeing is whole to be seen; E:HE dyeing; F:Masson dyeing.
The specific embodiment
Below in conjunction with accompanying drawing the specific embodiment provided by the invention is elaborated.
The preparation (one) of embodiment 1 engineering blood vessel of the present invention
1 material and method
1.1 the preparation of gelatin/polycaprolactone electrospinning film
Take by weighing the 0.5g gelatin and the 0.5g polycaprolactone is dissolved in the trifluoroethanol of 10ml with electronic analytical balance, stir 24h to dissolving fully, obtaining concentration is the 10%(grams per milliliter) gelatin/polycaprolactone spinning liquid, add 10 μ l acetic acid in the solution, stir 10-20min and become clarification by muddiness to solution.Select the syringe of 10ml for use, 1.2mm the syringe needle of internal diameter, extract gelatin/polycaprolactone spinning liquid, be fixed on the electrostatic spinning apparatus, the adjustment electrostatic pressure is 10kv, accepting distance is 12cm, injection rate is 2ml/h, carries out electrospinning, and the employing aluminium foil is receiving device, spinning 3h obtains gelatin/polycaprolactone composite nano-fiber membrane.When making up blood vessel, be cut into rectangle sticks wide 2 centimetres, long 12 centimetres, it carried out vacuum lyophilization disinfect back standby.
1.2 human smooth muscle cell is cultivated
Get human umbilical artery, form tissue in superclean bench supernatant expel stagnation, divest tunica adventitia of artery, vertically cut blood vessel open, passivity is struck off lining endothelium, removes endotheliocyte, residue tunica media tissue.After the normal saline that contains penicillin and streptomycin washes repeatedly, cut into the piece of tissue of 1mm * 1mm size repeatedly with the ophthalmology curved scissors, and will shear the good fritter of organizing and evenly put at the bottom of bottle piece of tissue spacing 0.5cm.Build bottle cap, the culture bottle that overturns gently injects the culture fluid that an amount of low sugar DMEM (Hyclone, the U.S.) contains 10% hyclone (FBS) (Hyclone, the U.S.) up and in bottle at the bottom of allowing bottle, places to contain 5% CO
237 ℃ of incubators in place 2-4h make piece of tissue dry and attach with the bottle wall after, culture bottle slowly overturn to keep flat, and piece of tissue is immersed in the culture fluid fully, continues to leave standstill to cultivate 3-5d.Treated that cell changes liquid on every side after piece of tissue is swum out of, treat to go down to posterity after 90% fusion.
1.3 scanning electron microscopic observation
Smooth muscle cell is collected in digestion, and be planted on a slice gelatin/polycaprolactone electrospinning film, take out complex behind the In vitro culture 1d, fix through 2.5% glutaraldehyde, ethanol gradient dehydrate then, ion sprays the instrument metal spraying, carries out scanning electron microscopic observation and takes pictures, and organizes in contrast with simple gelatin/polycaprolactone electrospinning membrane material group.
" 1.4 egg roll " method tissue engineering vessel
With wide 2 centimetres, long 12 centimetres of disinfecting the rectangle gelatin/polycaprolactone electrospinning film is tiled on the culture dish standby, collect the smooth muscle cell of cultivating with 0.25% trypsinization, it is suspended in the low sugar DMEM culture fluid of 10% FBS again, and cell concentration is 10 * 10
6/ ml, inoculation 1ml cell suspension makes cell suspension be paved with whole electrospinning film on gelatin/polycaprolactone electrospinning film, is positioned over to contain 5% CO
237 ℃ of incubators cultivate, behind 30min, add a little culture fluid in material surface, spend the night behind culture dish until adding the 10ml culture fluid behind the 2h.Second day is that PVC catheter " egg roll " method of 6mm is curled into tubulose (number of turns is about: 12cm/ (6 * 3.14)=6.4 circle) with cell material composite electrospun film along diameter, and put into the 50ml centrifuge tube after leaving standstill half an hour, add low sugar DMEM, the culture fluid of 10% FBS covers complex, places to contain 5% CO
237 ℃ of incubators cultivate.Change liquid every 2 days half amounts, cultivate 1 all backs nude mice subcutaneous transplantation, draw materials after 8 weeks.The building process of above-mentioned engineering blood vessel can be referring to Fig. 1 and Fig. 2.
1.5 the evaluation of the engineering blood vessel that makes up
1.5.1 histology
HE dyeing: with haematoxylin dyeing 5min after the engineering blood vessel section dewaxing aquation that makes up, the flushing back hydrochloride alcohol differentiation several seconds, blue 30min is returned in washing from the beginning, ethanol dehydration behind Yihong dyeing 1min, the transparent back of dimethylbenzene resinene mounting.
Masson dyeing: with washing again after the plain 5-10min washing of the WeigerShi Garapa back 1% hydrochloride alcohol differentiation after the engineering blood vessel section dewaxing aquation that makes up, Ponceaux acid fuchsin liquid dyes 5-10min, washing back 1% phosphomolybdic acid aqueous solution is handled 5min, green liquor is redyed 5min, 1% glacial acetic acid is handled 1min, 95% dehydration of alcohol repeatedly, the anhydrous alcohol dehydration, dimethylbenzene is transparent, the neutral gum mounting.
1.5.2 biomechanics detects
Cut off after the engineering blood vessel that makes up is cut into the wide ring-type of 3mm, with detecting its mechanical property by omnipotent pulling force detector (Instron-3343) behind its thickness of miking, the calculating elastic modulus.Getting the little porcine aorta that internal diameter is similarly 6mm organizes in contrast.
2 results
2.1 the evaluation of gelatin/polycaprolactone electrospinning film
The gelatin that the static spinning method prepares/polycaprolactone electrospinning membrane material has certain mechanical strength (fracture strength 2.74 ± 0.20MPa, elastic modelling quantity 33.43 ± 3.07 Mpa, strain 1.77 ± 0.15mm/mm) during fracture, operability more intense (Fig. 3 A).The scanning electron microscope result of simple gelatin/polycaprolactone electrospinning membrane material shows that nanofiber is even, smooth, continuous, the bionical preferably fibre fineness (Fig. 3 C) of n cell epimatrix collagen.With the compound back of human smooth muscular cells In vitro culture 1d, the visible smooth muscle cell of scanning electron microscope adheres to this material surface, and cell is sprawled well (Fig. 3 D).
The outer tissue engineering vessel of " 2.2 egg roll " body of laws
The outer tissue engineering vessel of " egg roll " body of laws can be referring to Fig. 1 and Fig. 2.Earlier gelatin/polycaprolactone the material that disinfects is tiled in (Fig. 2 A) in the big culture dish, collector's umbilical artery smooth muscle cell is inoculated in spend the night behind gelatin/polycaprolactone electrospinning film surface (Fig. 2 B) uniformly, gelatin/polycaprolactone electrospinning the film that to inoculate cell in second day is wrapped on the PVC catheter, final engineering blood vessel (Fig. 2 C that forms tubulose, 2D), it is subcutaneous that In vitro culture is transplanted to nude mice after 1 week then, makes it further ripe.
2.3 the detection of the engineering blood vessel that makes up
The nude mice subcutaneous transplantation was drawn materials after 8 weeks, saw substantially: the boundary of the engineering blood vessel of structure and nude mice autologous tissue is clear, good springiness, hardness is moderate, inner wall smooth, but keep flat independent support become tubulose (Fig. 4 A, 4B, 4C).Evenly (Fig. 4 D, 4E), Masson dyeing shows collagen fiber abundant (Fig. 4 F) in the engineering blood vessel that makes up for HE section display organization compact structure, cell distribution.
The preparation (two) of embodiment 2 engineering blood vessels of the present invention
Preparation method is with embodiment 1, and difference only is: the rectangle gelatin/polycaprolactone electrospinning film of preparation is wide 2 centimetres, long 13 centimetres; In the step 1.4, cell material composite electrospun film is that PVC catheter " egg roll " method of 6mm is curled into tubulose along diameter, and then the number of turns of Juan Quing is: 13cm/ (6 * 3.14)=6.9 circle.The result: the engineering blood vessel of preparation is the same with embodiment 1, and is clear with nude mice autologous tissue boundary, good springiness, and hardness is moderate, and inner wall smooth becomes tubulose but keep flat independent support; HE section display organization compact structure, cell distribution is even, and Masson dyeing shows that collagen fiber are abundant in the engineering blood vessel that makes up.
Comparative Examples 1
Preparation method is with embodiment 1, and difference only is: the rectangle gelatin/polycaprolactone electrospinning film of preparation is wide 2 centimetres, long 11 centimetres; In the step 1.4, cell material composite electrospun film is that PVC catheter " egg roll " method of 6mm is curled into tubulose along diameter, and then the number of turns of Juan Quing is: 11cm/ (6 * 3.14)=5.8 circle.Result: the engineering blood vessel mechanical property deficiency of preparation.
Comparative Examples 2
Preparation method is with embodiment 1, and difference only is: the rectangle gelatin/polycaprolactone electrospinning film of preparation is wide 2 centimetres, long 14 centimetres; In the step 1.4, cell material composite electrospun film is that PVC catheter " egg roll " method of 6mm is curled into tubulose along diameter, and then the number of turns of Juan Quing is: 14cm/ (6 * 3.14)=7.4 circle.The result: there are the phenomena of mortality in the cell of the engineering blood vessel inside of preparation.
Comparative Examples 3
Preparation method is with embodiment 1, and difference only is: cell concentration is 8 * 10
6/ ml.The result: the engineering blood vessel of preparation fails to form favorable tissue.
Comparative Examples 4
Preparation method is with embodiment 1, and difference only is: the time of engineering blood vessel In vitro culture is 2 days half.The result: the engineering blood vessel undercapacity of preparation, easily loose.
Comparative Examples 5
Preparation method is with embodiment 1, and difference only is: the time of engineering blood vessel In vitro culture is 7 days half.The result: there are the phenomena of mortality in the cell of the engineering blood vessel inside of preparation.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.
Claims (8)
1. an engineering blood vessel is support with tubulose gelatin/polycaprolactone electrospinning film, it is characterized in that smooth muscle cell is uniformly distributed in the inside of described support.
2. engineering blood vessel according to claim 1 is characterized in that, it prepares in accordance with the following methods:
A) gelatin/polycaprolactone material that disinfects is tiled in the vessel;
B) collect the umbilical artery smooth muscle cell and be inoculated in overnight incubation behind gelatin/polycaprolactone electrospinning film surface uniformly, form cell material composite electrospun film;
C) cell material composite electrospun film is wrapped on the pvc pipe cultivates, finally form the engineering blood vessel of tubulose.
3. engineering blood vessel according to claim 2 is characterized in that, the inoculative proportion of umbilical artery smooth muscle cell and gelatin in the step b)/polycaprolactone electrospinning film is 10 * 10
6Individual cell/(the electrospinning film of 2cm * 12cm); The number of turns that described cell material composite electrospun film is wrapped on the pvc pipe is the 6-7 circle, and cultivating for 1 week then can be for the engineering blood vessel of transplanting to form.
4. engineering blood vessel according to claim 3 is characterized in that, changes liquid once every 2 days half amounts in the process in described 1 week of cultivation.
5. the preparation method of the arbitrary described engineering blood vessel of claim 1-4 is characterized in that, may further comprise the steps:
A) gelatin/polycaprolactone material that disinfects is tiled in the vessel;
B) collect the umbilical artery smooth muscle cell and be inoculated in overnight incubation behind gelatin/polycaprolactone electrospinning film surface uniformly, form cell material composite electrospun film;
C) cell material composite electrospun film is wrapped on the pvc pipe cultivates, finally form the engineering blood vessel of tubulose.
6. preparation method according to claim 5 is characterized in that, the inoculative proportion of umbilical artery smooth muscle cell and gelatin in the step b)/polycaprolactone electrospinning film is 10 * 10
6Individual cell/(the electrospinning film of 2cm * 12cm); The number of turns that described cell material composite electrospun film is wrapped on the pvc pipe is the 6-7 circle, cultivates for 1 week then to form for the engineering blood vessel of transplanting.
7. preparation method according to claim 6 is characterized in that, changes liquid once every 2 days half amounts in the process in described 1 week of cultivation.
8. the arbitrary described engineering blood vessel of claim 1-4 is in the purposes of preparation among the artificial blood vessel.
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| CN104287869A (en) * | 2014-09-19 | 2015-01-21 | 上海市肺科医院 | Novel nanofiber membrane and yarn support for trachea transplantation and method for manufacturing novel nanofiber membrane and yarn support |
| CN105055060A (en) * | 2015-08-04 | 2015-11-18 | 上海交通大学医学院附属上海儿童医学中心 | Tracheal stent and application thereof |
| CN105695401A (en) * | 2016-03-29 | 2016-06-22 | 南京大学医学院附属鼓楼医院 | Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells |
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| CN110960727A (en) * | 2019-12-19 | 2020-04-07 | 广州市妇女儿童医疗中心 | Tissue-engineered urethral stent graft and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110960727A (en) * | 2019-12-19 | 2020-04-07 | 广州市妇女儿童医疗中心 | Tissue-engineered urethral stent graft and preparation method and application thereof |
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