CN101259292A - Construction method of tissue engineering blood vessel - Google Patents
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- CN101259292A CN101259292A CNA2007100642107A CN200710064210A CN101259292A CN 101259292 A CN101259292 A CN 101259292A CN A2007100642107 A CNA2007100642107 A CN A2007100642107A CN 200710064210 A CN200710064210 A CN 200710064210A CN 101259292 A CN101259292 A CN 101259292A
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Abstract
The invention relates to a construction method of a tissue engineering blood vessel, belonging to the field of tissue engineering. A method for constructing a tissue engineering blood vessel comprises the following steps: (1) connecting the tubular autologous living tissue pedestal material in a bioreactor for continuous perfusion under the in vitro aseptic condition, and storing culture solution outside a tube cavity; (2) suspending autologous endothelial cells obtained by in vitro culture with M199, or suspending autologous endothelial-like cells generated by bone marrow mononuclear cell induction with endothelial progenitor cell induction culture solution, and inoculating on the inner surface of the lumen according to a high-density planting method; (3) and (3) after endothelial cells or endothelial-like cells are adhered, communicating the intra-cavity circulation and the external circulation, simulating blood circulation to give appropriate pressure stimulation and shear force stimulation, and culturing in a carbon dioxide incubator for 5-7 days. The invention has the advantages that: has perfect cell compatibility and enough mechanical strength, overcomes the defects of immunological rejection, inflammatory reaction and the like caused by heterologous materials, and has short preparation time.
Description
Technical field
The present invention relates to a kind of construction method of engineering blood vessel, a kind of method from body living tissue material external structure blood vessel graft of more specifically saying so belongs to field of tissue engineering technology.
Background technology
At present, cardiovascular disease has become the operating method displacement of the finally necessary dependence of one of disease of serious harm human health, particularly vascular occlusive disease lesion vessels.Vascular graft at present commonly used comprises the artificial blood vessel that makes with nondegradable synthetic material and the of the same race or heterogenous blood vessel handled through distinct methods and from the body blood vessel.The subject matter of artificial blood vessel's material is to have thrombosis, blood coagulation problem; Mainly there are problems such as rejection, calcification, poor durability in of the same race or heterogenous blood vessel.Remain the main source of medium and small vascular graft at present from the body blood vessel, but it is limited from body blood vessel source, limited amount, particularly among gerontal patient, renal disease patient, diabetes, the hyperpietic, the more apparent rareness of transplantable no pathological changes blood vessel, especially in many bypasses of needs or in surgical patient, all the more so.Though, do not obtain breakthrough at present yet through effort for many years.
In recent years, the proposition of tissue engineering method, be expected to address these problems completely, its principle is the method for application cell plantation, patient's living cells is become activated tissue in vitro cultivation, be implanted in the patient body, living cells secretory cell epimatrix composition is transformed gradually, finally realizes the regeneration of living tissue organ again.
Traditional tissue engineering technique is a support with degradable macromolecular material, but there is poor mechanical property in macromolecular material, is difficult to moulding, catabolite and has in various degree shortcomings such as toxicity in the part.Therefore, the material of biogenetic derivation more and more comes into one's own.With the collagen pedestal is reconstitution cell extracellular matrix materials incomparable advantage of tool traditional material on biocompatibility of representative, yet the problem that exists at aspects such as mechanical property, compliances has restricted this extensive use of class material in organizational project greatly.The cell material that takes off in xenogenesis or allosome source exists distinctive advantage on mechanical strength, compliance, but has problems such as communicate illness, antigen be residual simultaneously.In addition, tissue calcification has become the major reason that this type of material of restriction is used.In recent years, the organization material in receptor autophosphorylation source has caused people's attention with its unique advantage.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of with the method from body living tissue material construction engineering blood vessel graft materials, can make up the engineering blood vessel graft that is suitable for as vascular graft in this method of external application.
For achieving the above object, the present invention's (see figure 1) by the following technical solutions:
A kind of construction method of engineering blood vessel comprises the steps:
(1) tubulose is connected in the bioreactor of continous perfusion the full culture fluid of the outer storage of tube chamber under external aseptic condition from body living tissue pedestal material; Blood vessel is cultivated the ecological simulation system and is divided internal recycle and outer circulation, is driven by more than two grades of adjustable peristaltic pumps respectively.Reactor reference literature design (see figure 2) is (referring to Mall JW, Philipp AW, RademacherA, et al.Re-endothelialization of punctured ePTFE graft:an in vitro study under pulsedperfusion condition.Nephrol Dial Transplant, 2004,19:61-67.).Interior outer circulation full respectively the internal recycle culture fluid and the outer circulation culture fluid of configuration voluntarily.The liquid-accumulating bottle of (A) internal recycle among the figure; (B) peristaltic pump of internal recycle, the liquid stream direction is as shown by arrows; (C) liquid of internal recycle holds; (D) perfusate chamber of reactor; (E) peristaltic pump of outer circulation; (F) liquid-accumulating bottle of outer circulation.A1, a2 are respectively the gas exchange air intake duct of internal recycle and outer circulation, and b1, b2 are respectively the gas exchange exhaust duct of internal recycle and outer circulation, all are connected to the pin type micropore filter of aperture less than 0.2 μ m, filtration sterilization.
What (2) In vitro culture is obtained suspends with M199 from the body endotheliocyte, or BMNC is induced suspending with endothelial progenitor cells inducing culture liquid from the body endothelioid cells of generation, is inoculated in the tube chamber inner surface by the high-density planting method;
(3) treat that endotheliocyte or endothelioid cells stick back on-tube intracavity circulation and outer circulation, and the simulate blood circulation gives suitable shearing force stimulation (5～15dyn/cm2) gradually, pressure 95/55mmHg, in 37 ℃ of 5% CO2 gas incubator, cultivated 5～7 days, obtain engineering blood vessel sample complex.
Described construction method from body living tissue pedestal material: laboratory animal abdominal cavity or subcutaneous is gone in the medical silicone tube heeling-in that will be fit to length with the mini-invasive incision technology, as the foreign body model, after 2～3 weeks, the complete taking-up of silica gel tube pod capsule that will be wrapped to form from the body living tissue with the method for aseptic operation.After wiping out the two ends of sealing pod capsule, behind the extraction silica gel tube, form tubulose from body living tissue structure, as the pedestal material of engineering blood vessel structure.
The present invention at first implants with foreign body, induces body to produce encapsulation reaction, to make up the blood vessel pedestal material that can use for organizational project (in general flesh pipeline and capsule sexual organ's structure all suit to use the method).Cultivate at amplification in vitro simultaneously and induce to the endothelium direction, as seed cell from the body vascular endothelial cell or with the mesenchymal cell of derived from bone marrow.Under static state or rotating condition, seed cell is inoculated in the inner surface of blood vessel pedestal material then.Then under external continous perfusion situation, give the pulsation and the stimulation of beating, on the basis that guarantees the survival of pedestal material, promote the ripe and differentiation of living cells.Final formation can reach the purpose that the application organizes engineering makes up vascular graft for the blood vessel sample graft materials of transplanting.
We follow up a case by regular visits to after 1 month to be the femoral artery that engineering blood vessel graft materials that pedestal makes up is transplanted to cell donor from the body living tissue, do not have and obviously block and thrombosis, and the histology finds that inner membrance does not have obvious hypertrophy performance.
Advantage of the present invention is: attempted first with from the body living tissue as organizational project pedestal material construction organizational project organ, guaranteeing on the basis of body living tissue material survival, to have carried out the research of cell seeding at material surface.Living tissue pedestal pipe from the body source has perfect cell compatibility and enough mechanical strengths, and constructed organ can be got rid of the risk of xenogenesis, the generation of allosome immunological rejection fully, has overcome the heterologous material and has caused defectives such as immunologic rejection and inflammatory reaction.Time required for the present invention is shorter, well below 3～6 months cycle of similar technology required time.
The invention will be further described below in conjunction with the specific embodiment; it is not limitation of the invention; according to prior art well known in the art; embodiments of the present invention are not limited to this; therefore all this areas of having done according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the sketch map of ultimate principle of the present invention, is connected in the vitro reactions device with planting in what intraperitoneal obtained from the biological pipe of body living tissue, in tube chamber face plantation endotheliocyte, forms similar capillary structure.
Fig. 2 is the sketch map of reactor used in the present invention and blood vessel biosimulation system.
Fig. 3 is from 400 times of common light microscopic photos of the HE of the biological pipe of body living tissue dyeing.
Fig. 4 is through 400 times of ordinary optical microscope photos of the HE of the engineered blood vessel complex of the present invention's structure dyeing.
Fig. 5 be behind the repopulating cell from the painted 200 times of ordinary optical microscope photos of body living tissue biological pipe Masson.
Fig. 6 is the painted 200 times of ordinary optical microscope photos of the Masson of natural blood vessel.
Fig. 7 is the painted 200 times of ordinary optical microscope photos of orcein method elastic fibers through the engineered blood vessel complex of the inventive method structure.
Fig. 8 is the painted 200 times of ordinary optical microscope photos of the elastic fibers of natural artery blood vessel.
Fig. 9 is fresh 200 times of stereoscan photograph from the biological tube-surface structure of body living tissue before the repopulating cell not.As seen a large amount of fibre structures and extracellular matrix components, accidental hemocyte and fibroblast projection.
Figure 10 is 200 times of stereoscan photograph of rotation high-density method inoculation endothelium tube chamber inner surface after 6 hours.
Figure 11 is amplified to 1000 times stereoscan photograph for Figure 10.As seen a large amount of cell adhesions of tube chamber inner surface, most cells are still rounded, and the part cell stretches to stick and is fusiformis.
Figure 12 is 200 times of stereoscan photograph of static plantation tube chamber inner surface after 24 hours.
Figure 13 is amplified to 1000 times stereoscan photograph for Figure 12.As seen the adherent cell adherent growth stretches, and there is the certain utmost point tropism part, but overall polarity is not obvious.
Figure 14 is after giving cumulative shearing force stimulation, plants 200 times of stereoscan photograph of tube chamber inner surface after 72 hours.
Figure 15 is amplified to 1000 times stereoscan photograph for Figure 14.The tropism is obvious for the visible cell growth utmost point, and the cell major axis is generally arranged along the liquid stream direction, and confluent growth trend is arranged between visible cell.
When Figure 16 reaches 5 days for the dynamic perfusion cultivation, 200 times of stereoscan photograph of tube chamber inner surface.
Figure 17 is amplified to 1000 times stereoscan photograph for Figure 16.The tropism is obvious for the visible cell utmost point, and cell is arranged closely, and the tube chamber inner surface is smooth, and the cell growth converges.
Figure 18 is 200 times of stereoscan photograph of natural dog femoral artery intravascular space face.
Figure 19 is amplified to 1000 times stereoscan photograph for Figure 18.The visible cell confluent growth, smooth surface, it is obvious that cell is arranged utmost point tropism.
Figure 20 is smooth muscle specific actin (the 400 times of ordinary optical microscope photos of immunohistochemical staining of SM-α-actin) of the engineered blood vessel sample complex of our bright structure.
Figure 21 is 400 times of ordinary optical microscope photos of the SM-α-actin immunohistochemical staining of natural femoral artery.
Figure 22 is 400 times of ordinary optical microscope photos of VIII factor related antigen immunohistochemical staining of the engineered blood vessel sample complex of our bright structure.
Figure 23 is 400 times of ordinary optical microscope photos of the VIII factor related antigen immunohistochemical staining of natural artery.
Figure 24 is behind the endothelial cell seeding with the PKH-26 labelling, 200 times of photos of the laser confocal microscope of intravascular space face.
Figure 25 is 400 times of photos of laser confocal microscope of Figure 24 specimen.
Figure 26 is 200 times of photos of cross section laser confocal microscope of Figure 24 specimen.
Figure 27 is 400 times of photos of the cross section laser confocal microscope of Figure 24 specimen.The cell seeding of visible fluorescence labelling is also sprawled and is grown in the tube chamber inner surface, is monolayer alignment.
Figure 28 is 1000 times of stereoscan photograph of the inner surface after the biological pipe of body living tissue and platelet effect of repopulating cell not.Do not plant the visible significantly platelet adhesion reaction of tissue surface of endotheliocyte and assemble, be bulk, be difficult to differentiate single hematoblastic form.
Figure 29 is organizational project complex and the 1000 times of stereoscan photograph of the inner surface after the platelet effect behind the plantation endotheliocyte.The organizational project composite surface of plantation endotheliocyte does not almost have obvious platelet adhesion reaction, and it is smooth that the surface still keeps.
Figure 30 is 6000 times of transmission electron microscope photos of the engineered blood vessel complex inner surface of the present invention's structure.As seen there is closely to be connected to form cell surface villous spline structure between endotheliocyte.
Figure 31 is 20,000 times of transmission electron microscope photos of the engineered blood vessel complex tube wall cell of the present invention's structure.
Figure 32 is 30,000 times of transmission electron microscope photos of the engineered blood vessel complex tube wall cell of the present invention's structure.The filament spline structure of visible bunchy and gulp down extracellular matrixs such as drink vesicle, cell surface lining proteoglycan in the visible a plurality of macula densas in visible cell film surface, the cell.
The specific embodiment
Embodiment 1.
One. reagent and material
1. phosphate buffer (PBS liquid): commercially available PBS powder (Gibco) parcel, be dissolved in the 1000ml distilled water, adjust pH 7.2, after the filtration sterilization, packing, put 4 ℃ standby.
2.M199 culture fluid: commercially available M199 powder (Gibco) pouch, add sodium bicarbonate 1.2g, 1.5mol/LHEPES 10ml, tri-distilled water adds to 1L, regulates pH to 7.2, and the 250ml packing is pressed in filtration sterilization, and it is standby to put 4 ℃ of storages.
3.DMEM culture fluid: commercially available DMEM powder (Gibco) pouch, add sodium bicarbonate 1.2g, 1.5mol/LHEPES 10ml, tri-distilled water adds to 1L, regulates pH to 7.2, and the 250ml packing is pressed in filtration sterilization, and it is standby to put 4 ℃ of storages.
4. endotheliocyte and internal recycle culture fluid: the M199 culture fluid that contains the endothelial cell growth fill-in (ECGS is available from Sino-Japanese Friendship Hospital) of 15～20%FBS and 0.1mg/ml.
5. outer circulation culture fluid: contain 20% hyclone (FBS, Gibco), 2ng/ml basic fibroblast growth factor (b-FGF, PeproTech), 50ng/ml platelet derived growth factor-BB (PDGF-BB, PeproTech), 10ng/ml insulin like growth factor (IGF, PeproTech), 0.5ng/ml epithelical cell growth factor (EGF, DMEM solution PeproTech).
6. endothelial progenitor cells inducing culture liquid: will add that by specification mixes under the EGM-2MV SingleQuots aseptic condition available from the EBM-2 of U.S. Canbrex bio science valkersville company, the rearmounted 4 ℃ of preservations of packing.
7.0.05% trypsin-0.53mmol/L EDTA.4Na solution: (Gibco) 0.5g trypsin 1: 250), EDTA.4Na 0.2g adds no Ca
2+, Mg
2+BSS adds to 1L, filtration sterilization, packing bottle ,-20 ℃ of preservations.
8. medical silicone tube is selected the medical hollow silica gel tube of diameter 4mm, cuts out the segment into length 2cm, 4cm, 6cm respectively, packs back oxirane disinfection sterilization, and placement 1 all backs are standby in the air at room temperature.
9. penicillin/streptomycin solution (100 times of concentration) penicillin 1.0106IU, streptomycin 1g adds 0.9% normal saline to 100ml, filtration sterilization, the packing bottle is put-20 ℃ of preservations.
10.0.1% collagenase solution: commercially available IV Collagen Type VI enzyme (Sigma) 100mg, add PBS solution to 100ml, filtration sterilization divides device-20 ℃ preservation.
Two. method:
(1) endotheliocyte In vitro culture
Under the aseptic operation condition, cut the about 10cm of laboratory animal external jugular vein vascular tissue, the collagenase intra-bladder instillation digestion back collecting cell with 0.1% is behind the centrifuge washing, with 1 * 10
6Individual/ml cell density is inoculated in advance the culture bottle with the gelatin shop fixtures, the M199 culture fluid that adds 20% hyclone, 0.1mg/ml vascular endothelial growth fill-in (ECGS), 200U/ml penicillin, 0.2ug/ml streptomycin, obedient walls are cultivated in 37 ℃ of 5%CO2 incubators, get second to six generation cell be used for experiment.Experimental cell is left and taken sample and is carried out the VIII factor, the evaluation of CD31 immunohistochemical staining.
The medulla mesenchyma cell is to the inducing culture and the discriminating of endothelium:
After the experimental animal anesthesia, aseptic condition extracts bone marrow 10ml down.After PBS dilution in 1: 1, be laid on density and be 1.073 lymphocyte separation medium surface, 2000 left the heart 20 minutes under the room temperature, draw the mononuclearcell layer, leave heart washing 5～10 minutes with PBS liquid dilution back 1500, suspend with the EBM-2EGM-2V culture fluid, counting is also adjusted cell density, by 5 * 10
5/ cm
2Be inoculated in advance in the culture bottle with fibronectin (Fn) shop fixtures, place 37 ℃ of 5%CO
2Cultivate in the incubator of saturated humidity, inducing the bone marrow stroma stem cell directed differentiation is endotheliocyte.Changed liquid once in per 2～3 days after the inoculated and cultured, observation of cell form under the phase contrast microscope, treat cell cover with bottle at the bottom of 80% o'clock with 0.05% trypsin-0.53mmol/L EDTA.4Na (Gibco) solution had digestive transfer culture.Second filial generation cell seeding carries out the VIII factor, CD31, the evaluation of FLK-1 immunohistochemical staining on coverslip.
(2) from the acquisition of body living tissue biomaterial
Behind the laboratory animal anesthesia, under the aseptic condition, make long 1～2cm otch, the laboratory animal intraperitoneal is gone in the medical silicone tube heeling-in that is fit to length or the back subcutaneous.After 2～3 weeks, equally with the complete taking-up of silica gel tube pod capsule of the method for Wicresoft with peritoneum or subcutaneous tissue parcel.In aseptic condition down suitably after the finishing, wipe out the two ends of sealing pod capsule after, extract silica gel tube out after, form tubular structure.
(3) from the In vitro culture and the cell seeding of body living tissue pedestal pipe
With being connected in the blood vessel bioreactor of external continous perfusion under with 3-0 silk thread aseptic condition of obtaining from body living tissue biomaterial pipe, the tube chamber exocoel is filled with outer circulation liquid, adopt the high-density planting method with suspending with the internal recycle culture fluid that In vitro culture obtains, by 5 * 10 from body jugular vein endotheliocyte
6/ ml concentration is inoculated in the developmental tube intracavity, (37 ℃ of 5%CO2) hatched 2～4 hours with manual upset or at the uniform velocity slow rotation in the carbon dioxide incubator, so that cell fully sticks, connect internal recycle and outer circulation then, at first static culture is 24 hours, and perfusion rate is regulated in simulate blood circulation then, gradually by being increased to 3～9ml/s, shearing force stimulates (5～15dyn/cm in 3 days
2), pressure 95/55mmHg.In CO2 gas incubator, continue to cultivate, promptly get engineering blood vessel to 5 days.
Embodiment 2. evaluation of result
To embodiment 1 relate to fresh in the biological pipe of body living tissue and engineering blood vessel specimen taken respectively, carry out mechanics according to purgation and measure and Biological Detection.
One. method
(1) mechanics detects
1. the mensuration of fracture tensile strength: specimen is cut into the sheet material shape, on puller system it is broken, the maximum pull of being surveyed is the tensile strength of material of specimen divided by the cross-sectional area of experiment material, and expression is from the stretch-proof ability of body living tissue material.
2. the mensuration of elongation at break: specimen is cut into the sheet material shape, on puller system it is broken, the record sample is from being stretched to the length variations of being broken, divided by the original length of sample, percentage ratio be the elongation at break of specimen material, expression distortion of materials ability.
3. the burst strength of specimen is measured: will be connected in the burst pressure meter from body living tissue pipe specimen, and the free-end sealing, full PBS liquid in the device is regulated pressurization bolt compression intracavity liquid, with the pressure that increases in the Pressure gauge record tubing.Burst pressure is defined as the maximum pressure that tube type material is reached before breaking, the tolerance that the expression material changes pressure.
4. the suture strength of specimen is measured: experimental specimen one end is clipped on the puller system, and the other end is connected on another anchor clamps of puller system with the nylon suture of 6-0.Then with the speed of 1mm/min tractive at the uniform velocity, the pulling force when record ruptures fully.The property sewed up of expression material.
(2) Biological Detection:
1. the basic structure of specimen: the neutral formalin fixed preparation with 10%, paraffin embedding is cut into the thin slice of 4 micron thickness, through dimethylbenzene dewaxing, serial dehydration of alcohol, haematoxylin-Yihong dyeing, observes the roughly cell of specimen and forms and arranged distribution.
2. the fibre structure of specimen: neutral formalin fixed preparation with 10%, paraffin embedding, be cut into the thin slice of 4 micron thickness,, observe the fiber alignment and the composition of specimen through dimethylbenzene dewaxing, serial dehydration of alcohol, Masson dyeing and the dyeing of orcein method elastic fibers.
3. the surface texture of specimen: specimen is with 2.5% glutaraldehyde and 1% osmic acid double fixed, and ethanol dewaters step by step, the carbon dioxide critical point drying, and the vacuum metal spraying, scanning electron microscope (SEM) is observed the ultrastructure of biological tube cavity face.
4. the immunohistochemical staining inspection of specimen: use the neutral formalin fixed preparation, paraffin embedding, be cut into the thin slice of 4 micron thickness, through dimethylbenzene dewaxing, serial dehydration of alcohol, antigen retrieval, by two step method add successively one anti-and two anti-after, drip chromogenic reagent, haematoxylin is redyed, and observes the expression of the specific VIII factor, α-actin.
5. the fluorescent labeling spike of endotheliocyte
With PKH26-GL preliminary making endotheliocyte, by 3 * 10
-6M dosage indicia 1 * 10
7Individual cell.Press preceding method with the endothelial cell seeding of labelling in surface from body living tissue blood vessel pedestal material.The glutaraldehyde of gained tub of tissue row 2.5% is fixed.To manage spline structure and be trimmed to lamellar, inner cavity surface places the fluorescence co-focusing microscopically to observe down, and shooting.Get the part specimen simultaneously, with the embedding of OCT glue ,-25 ℃ of sections in freezing 15 seconds of the liquid nitrogen, freezing microtome, thickness is 8 μ m, after the glycerol nial polish mounting, puts under the Laser Scanning Confocal Microscope and observes on the microscope slide.
6. platelet adhesion reaction test
Handle and the pedestal pipe of not planting endotheliocyte is contrast through the same terms to insert reactor, blood vessel sample analog static state behind platelet suspension and the plantation endothelium is hatched, take out after 2 hours, wash the tube chamber face 3 times with PBS, after 2.5% glutaraldehyde was fixing, row SEM observed.
7. the transmission electron microscope observing of engineering blood vessel
With the specimen that 2.5% glutaraldehyde fixedly spends the night, after PBS washed 3 times, 4 ℃ of mordant dyeings of 1% Osmic acid. are 30min fixedly, the dehydration of acetone series, the epoxy resin embedding, semithin section preparation, Toluidine blue staining, select region-of-interest, be cut into the ultrathin section of 8 nanometers, observe under the Electronic Speculum.This group test specimen all send the Capital University of Medical Sciences's attached Tiantan Hospital electronic microscopy room to finish.
Three. experimental result
(1) mechanics testing result
1. fracture tensile strength: fresh in body living tissue pipe hot strength 4.7 ± 2.3MPa, cultivating bleeding from anus pipe analog is 4.5 ± 1.8MPa, and statistics does not have significant difference.
2. elongation at break: fresh elongation at break from body living tissue pipe is 34.2 ± 8.3%, and cultivating bleeding from anus pipe analog is 33.1 ± 10.2%, the statistics no significant difference.
3. burst strength: fresh burst strength 1100 ± 187mmHg from body living tissue pipe, the engineering blood vessel complex behind the repopulating cell is 1070 ± 115mmHg, statistics does not have significant difference.
4. sew up tolerance intensity: the stitching tolerance intensity of new fresh and alive group of biological pipe is 2.5 ± 0.3N, and the engineering blood vessel complex behind the repopulating cell is 2.4 ± 0.7N, and statistics does not have significant difference.
(2) Biological Detection result:
1. basic structure: fresh common HE dyeing dyeing from the biological pipe of body living tissue shows by being the spindle cell that concentric circles arranges in a large number and constitutes that cell is spindle shape, and internal layer is thin than okioplast, visible a small amount of inflammatory cell infiltration (see figure 3) in the tube wall.The visible tube chamber inner surface of organizational project complex monolayer pinacocyte after the plantation covers, and tube wall also is made up of the spindle cell that a large amount of concentric circles are arranged, and inflammatory cell obviously reduces, only accidental (see figure 4).
2. fibre structure: the organizational project complex behind the repopulating cell is wavy arrangement, class concentric circular spline structure, visible smooth muscle like cell, similar to natural blood vessel structure (seeing Fig. 5,6) therebetween through the Masson visible collagen fiber that dye.Both compare as can be seen, and both extracellular matrix components all have a large amount of collagen fiber to constitute, and are the concentric circular sample and arrange, and collagen fiber become wavy arrangement, dye among the figure to be blueness; Visible cellularity between fiber, smooth muscle cell is dyed blue redness; But the visible down significantly continuous substrate membrane structure of Fig. 6 mesexine cell forms, pinkiness wire among the figure, and do not have this structure among Fig. 5.No obvious elastic plate spline structure (seeing Fig. 7,8) in the orcein method elastic fibers dyeing visible tissue engineering multiple tube.Both compare as can be seen, and no tangible elastic fibers plate spline structure forms among Fig. 7, and visible significantly elastic fibers plate spline structure among Fig. 8.
3. surface texture: fresh in the visible filamentary structure of the biological tube-surface of body living tissue, and a large amount of extracellular matrix components, accidental hemocyte composition and fibroblast projection (see figure 9).Organizational project composite surface behind the repopulating cell is covered by endotheliocyte, and cell is arranged with liquid stream direction rule, and prolong in time and gradually converge, smooth surface is with natural blood vessel similar (seeing Figure 10,11,12,13,14,15,16,17,18,19).
4. immunohistochemical staining: the engineering blood vessel complex behind the repopulating cell has and the similar structure of natural blood vessel, tube wall all contains a large amount of α-actin positive cell, inner cavity surface lining cell monolayer, VIII factor stained positive (seeing Figure 20,21,22,23).As can be seen, organizational project sample of blood pipe complex and natural blood vessel that the present invention makes up are similar, and the cell monolayer stained positive of inner cavity surface lining is pale brown color.
5. the fluorescent labeling spike of endotheliocyte shows: PKH26 spike immunofluorescence positive cells is coated on engineering blood vessel complex inner cavity surface, Laser Scanning Confocal Microscope shows that inner cavity surface lining cell converges each other, almost cover whole inner cavity surface, visible fluorescence labelling positive cells is the monolayer shape on the cross section, and limitation is coated on tube chamber inner surface (seeing Figure 24,25,26,27).
6. platelet adhesion reaction experiment: the organizational project composite surface behind the plantation endotheliocyte does not almost have obvious platelet adhesion reaction, and the not repopulating cell of in reactor, handling through similarity condition from the biological tube-surface of body living tissue, then visible significantly platelet adhesion reaction is assembled agglomerating, is difficult to differentiate single hematoblastic form (seeing Figure 28,29).
7. transmission electron microscope detects: the endotheliocyte of plantation is monolayer alignment in luminal surface, and iuntercellular has and is tightly linked, and there is microvillus spline structure (seeing Figure 30) on the visible cell surface.In subcutaneous tube wall middle layer cells have tangible smooth muscle spline structure, as the filamentary structure of bunchy, gulp down the extracellular matrix components such as proteoglycan (seeing Figure 31,32) of drink vesicle, cell surface lining.
Claims (5)
1. the construction method of an engineering blood vessel comprises the steps:
(1) tubulose is connected under external aseptic condition in the bioreactor of continous perfusion from body living tissue pedestal material, the full culture fluid of the outer storage of tube chamber, blood vessel is cultivated the ecological simulation system and is divided internal recycle and outer circulation;
What (2) In vitro culture is obtained suspends with M199 from the body endotheliocyte, or BMNC is induced suspending with endothelial progenitor cells inducing culture liquid from the body endothelioid cells of generation, is inoculated in the tube chamber inner surface by the high-density planting method;
(3) treat that endotheliocyte or endothelioid cells stick back on-tube intracavity circulation and outer circulation, and the simulate blood circulation, regulate perfusion rate, give suitable Pressure stimulation, shearing force stimulation, in CO2 gas incubator, cultivated 5～7 days, and obtained engineering blood vessel sample complex.
2. the construction method of a kind of engineering blood vessel according to claim 1, it is characterized in that: described construction method from body living tissue pedestal material is: laboratory animal abdominal cavity or subcutaneous is gone in the medical silicone tube heeling-in that will be fit to length with the mini-invasive incision technology, as the foreign body model, after 2～3 weeks, the complete taking-up of silica gel tube pod capsule that will be wrapped to form from the body living tissue with the method for aseptic operation; After wiping out the two ends of sealing pod capsule, behind the extraction silica gel tube, form tubulose from body living tissue structure, as the pedestal material of engineering blood vessel structure.
3. the construction method of a kind of engineering blood vessel according to claim 1, it is characterized in that: described pressure is 95/55mmHg.
4. the construction method of a kind of engineering blood vessel according to claim 1 is characterized in that: it is 5～15dyn/cm that described shearing force stimulates
2
5. according to the construction method of the described a kind of engineering blood vessel of claim 1, it is characterized in that: described adjusting perfusion rate is to be increased to 3～9ml/s in 3 days gradually.
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