CN106109054A - Large aperture parallel polycaprolactone electrospinning cotton is utilized to build autologous tissue's engineered blood vessels - Google Patents

Abstract

The present invention relates to a kind of large aperture parallel polycaprolactone (PCL) electrospinning cotton, belong to biomedical engineering technology field.The present invention also provides for tissue blood vessel engineering rack, engineering blood vessel prepared by this electrospinning cotton.Its advantage shows: (1) large aperture polycaprolactone electrospinning cotton has good mechanical property and cell compatibility, and beneficially cell is quickly grown into；(2) the engineering blood vessel histiocyte finally given and extracellular matrix derive from autologous, do not produce immunological rejection；(3) polycaprolactone electrospinning cotton fiber arranged in parallel guides cell and the arrangement of extracellular matrix directed annular, can simulate histology's structure of native blood vessels.

Description

Large aperture parallel polycaprolactone electrospinning cotton is utilized to build autologous tissue's engineered blood vessels

Technical field

The present invention relates to biomedical engineering technology field, specifically, be to utilize large aperture parallel polycaprolactone electrospinning Cotton structure autologous tissue engineered blood vessels.

Background technology

Existing several blood vessel transplantation substitute remain not there is growth or allosome tissue to be easily caused immunologic rejection anti- Defect should be waited.Therefore, build and possess growth, and have the novel tissue engineered blood vessels of autologous tissue or cell to have important meaning Justice.

Usually, tissue engineering vessel is firstly the need of solving three problems: (1) organization bracket, and (2) seed cell comes Source and (3) tissue construction method." organization bracket " be cell rely adhere to and growth environment.Owing to artificial constructed is organized in The formation initial stage still lacks the extracellular matrix implanting emiocytosis, it is therefore desirable to provide the support of attachment artificially for cell；From On the other hand saying, organization bracket also determines final form, the selection of seed cell and the field planting method of constructed tissue, because of This, preparing suitable organization bracket is the key that engineering blood vessel successfully constructs.

In various scaffold for vascular tissue engineering preparation methoies, the more commonly used Static Spinning technology that utilizes is prepared Fibrous framework.By parent's cell, biodegradable macromolecule polymeric material, such as polycaprolactone (PCL), polylactic acid (PLA) etc. is joined Make certain density solution, load and connect the syringe having Micropump.From the polymer solution of syringe needle extrusion at electric field Filament can be pulled under the effect of power, and be gathered in reception device in some way, finally give electrostatic spinning fiber support.

Before, inventor is explored with regard to electrostatic spinning fiber film application in tissue engineering vessel, and builds Having found a kind of engineering blood vessel construction method: i.e. with smooth muscle, a kind of cell being widely present in big-and-middle tunica media layer is made For seed cell；Smooth muscle cell is planted in the most in advance on nanofiber electrospinning film, treats that cell is raw in its surface adhesion After length, curling is wound in cylindrical tube, then is transplanted under animal skins form vessel-like tissue.Due to the method similar " sandwich " Manufacturing process, is therefore also referred to as " sandwich method ".

The reason why using the building mode of this " the external plantation of seed cell-curling parcel-subcutaneous transplantation " is, The nanoscale electrostatic spinning fiber diameter that we had previously developed is tiny, the fine and close consolidation of arrangement between fiber, the most direct subcutaneous shifting Plant or after blood vessel orthotopic transplantation the autogenous cell electrospinning film that is difficult to grow into internal and realize systematism this and the Gore-of electrospinning film Result after Tex pipeline or the transplanting of bovine jugular vein pipeline is without the biggest difference.But, this also brings other problem: (1) seed The process that cells in vitro separated, cultivated amplification is complicated, and cell contamination, aging easily occurs；(2) combine " sandwich " will be loaded with After seed cell electrospinning film roll Qu Yizhi, seed cell, firstly the need of overcoming subcutaneous malnourished environment, this results in structure Low success rate of problem；(3) external repopulating cell is also easy to occur cell to run off.Therefore, utilize above-mentioned " sandwich " more difficult Construct satisfied engineering blood vessel.Fig. 1 illustrates an example " sandwich " engineering blood vessel and builds failed case, can See layering, the most organized material.Accordingly, it would be desirable to fundamentally improve the character of Static Spinning support.

It is within the contemplation of the invention that solve the traditional method spun electrostatic spinning nano fiber fine and closely woven consolidation of film, what cell was difficult to grow into asks Topic.Large aperture, crude fibre PCL electrospinning cotton and the application in autologous tissue's engineered blood vessels thereof with PCL as base stock are current Have not been reported.

Summary of the invention

It is an object of the invention to for the deficiency in existing electrostatic spinning fiber film (i.e. tradition " electrospinning film ") technology, it is provided that one Plant electrospinning cotton.

Another purpose of the present invention is to provide the application that a kind of electrospinning is cotton.

Another the purpose of the present invention is to provide the application of a kind of tissue engineering bracket.

For achieving the above object, the present invention adopts the technical scheme that: a kind of electrospinning for preparing engineering blood vessel Cotton, described electrospinning cotton is that polycaprolactone electrospinning is cotton.

Described electrospinning cotton is that large aperture parallel polycaprolactone electrospinning is cotton, and the aperture of electrospinning cotton is 20 μm～100 μm.

Described electrospinning cotton is the electrospinning that obtained by electrostatic spinning of the trifluoroethanol solution of polycaprolactone of 30～40% Cotton.

Described electrospinning cotton prepares by the following method: configuration quality volume ratio is the trifluoroethanol of the polycaprolactone of 35% Solution, then carries out electrostatic spinning with the cylinder of hollow out for collection device, obtains electrospinning cotton under the conditions of short distance reception.

For realizing above-mentioned second purpose, the present invention adopts the technical scheme that: described electrospinning is cotton in preparation tissue work The application of the support of journey blood vessel.

A kind of scaffold for vascular tissue engineering, described scaffold for vascular tissue engineering is that the tubulose utilizing electrospinning cotton to build props up Frame.

Described electrospinning cotton is that large aperture parallel polycaprolactone electrospinning is cotton.

The preparation method of described scaffold for vascular tissue engineering is: with cylinder as inner support, by large aperture polycaprolactone Electrospinning cotton is paperwrapped in periphery in the same direction, makes scaffold for vascular tissue engineering.

The preparation method of described scaffold for vascular tissue engineering is: configuration quality volume ratio is the three of the polycaprolactone of 35% Fluoroethanol solution, then carries out electrostatic spinning with hollow out four skeleton cylinder for collection device and obtains electrospinning cotton, with plantation rubber column jecket For inner support, electrospinning cotton is paperwrapped in rubber cylinder tube-surface in the same direction, makes scaffold for vascular tissue engineering.

For realizing above-mentioned 3rd purpose, the present invention adopts the technical scheme that: described scaffold for vascular tissue engineering exists Prepare the application in engineering blood vessel.

The invention has the advantages that:

1, large aperture polycaprolactone electrospinning cotton has good mechanical property and cell compatibility, and beneficially cell is quick Grow into.

2, the engineering blood vessel histiocyte finally given and extracellular matrix derive from autologous, do not produce immunologic rejection Reaction.

3, polycaprolactone electrospinning cotton fiber arranged in parallel guides cell and the arrangement of extracellular matrix directed annular, can simulate Histology's structure of native blood vessels.

Accompanying drawing explanation

Accompanying drawing 1: with tradition electrospinning film as timbering material, by " sandwich " method tissue engineering vessel.Utilize gelatin/ PCL (50:50, m/m) nanofiber electrospinning film is as intravascular stent, with mesenchymal stem cells MSCs as seed cell, in vitro Mesenchymal stem cells MSCs is planted in electrospinning film, and curling is wound in cylindrical tube (" sandwich "), then be transplanted to nude mice Subcutaneous tissue engineering vessel.Seen from drawing materials after transplanting 9 months still, gelatin/PCL electrospinning film (B) of stacking, shows electrospinning Film is undegraded, organize the formation of difference.Tissue slice HE dyeing (A, C and D) visible 6 layers of electrospinning film are separated from each other, middle almost without Cell is grown into.Outermost electrospinning film has a small amount of cell to grow into (figure A black asterisk marked region) at two symmetrical positions, point Not for subcutaneous to be close to skin and to be close to the region of body wall.Owing to these position blood supplies are sufficient, promote cell and grow into and part shifting Plant the survival of cell.C figure amplifies display cell and grows into the region (corresponding A figure solid line boxes position) of electrospinning film；D figure amplifies display Electrospinning theca cell is grown into juncture area (corresponding A figure dashed rectangle region).E figure be cell grow into electrospinning diaphragm area Masson dye Color, the collagen fiber of rarely seen lower floor blue dye on a small quantity.The tissue surface that outer layer indigo plant dye position does not eliminates when being and draw materials loosens connective group Knit.The seed cell core still remained under red arrow instruction second layer electrospinning film.Scale: A=1.0mm；C-E=100 μm.

Accompanying drawing 2: the preparation of Static Spinning PCL fibrous membrane (cotton) and form thereof.10%PCL Electrospun surface is pasted with aluminium foil Drum apparatus receive finally give fine and closely woven Electrospun (A).Between Electrospun, close-packed arrays becomes PCL electrospinning film (B).Printing opacity is visible The structure of the fine and closely woven consolidation of PCL electrospinning film, naked eyes are difficult to observe by fiber quality (C).35%PCL Electrospun hollow out device receives The crude fibre (D) that rear available oriented is consistent.After this fiber multiple-layer stacked, the PCL electrospinning of available short texture is cotton (E) crude fibre (F), aligned seen from printing opacity.

Accompanying drawing 3:PCL electrospinning film (cotton) sem observation.Tradition PCL nanofiber electrospinning film (comparison) and greatly Cotton (present invention) scanning electron microscope diagram of aperture PCL electrospinning.Under the conditions of identical enlargement ratio, PCL electrospinning membrane fiber is tiny, The hole reserved between fiber is the least (A, B)；And PCL electrospinning cotton fiber is thick, the hole formed between fiber is big (B, D).Scale: A, C=100 μm；B, D=10 μm.

Accompanying drawing 4: the arrangement of electrostatic spinning fiber and Diameter Distribution.Application ImageJ image analysis software measures Electronic Speculum figure The angulation of 150 fiber opposite planar coordinate x-axis and fibre diameter (A, B) in sheet.PCL's nanofiber electrospinning film (comparison) is flat All diameter about 0.67 ± 0.34 μm, and diameter about 3.90 ± 1.85 μm of large aperture PCL electrospinning cotton (present invention), its fibre diameter Chart of frequency distribution be respectively C and D.

The mechanical property of accompanying drawing 5:PCL electrospinning film (cotton).Material has different shapes when receiving pulling force in different directions State feature, it may have (consolidation PCL electrospinning film: A and B, large aperture PCL electrospinning is cotton: C and D) for different stretchable length.Use pulling force The stressing conditions when receiving pulling force condition of material measured by machine, and stress MPa in its unit are is unit, relative measurement Before original length change percent represent, obtain bi-material drawing along machine direction (E) and vertical fibers direction (F) Power-stretch ratio curve.Young's modulus is to evaluate the index of material compliance, i.e. linear ascent stage on stress-length increased curve Slope.It is (right that PCL electrospinning cotton (present invention) Young's modulus in the two directions in large aperture is all significantly less than tradition PCL electrospinning film According to) (G and H, * p < 0.05.).

The hydrophilic angle detection of accompanying drawing 6:PCL electrospinning film (cotton).By 5 μ L water droplets in electrospinning film (cotton), and detect water droplet respectively Hydrophilic angle on material difference fiber extension direction.Dropping water droplet after 50s water droplet in material become hydrophilic angle be shown in figure A～ D, often in figure, the upper right corner is parallel with material fiber direction for detection direction mode figure: A, C display detection camera；B, D display material Vertical with machine direction.On every kind of material different directions, hydrophilic angle formed by 0s, 20s and 50s is summarized in figure E (* p < 0.05).

Accompanying drawing 7: human smooth muscular cells growth on PCL electrospinning film (cotton).Human smooth muscular cells is (right at PCL electrospinning film According to, A) and cotton (present invention, the B) In vitro culture cellular morphology after 6 days of PCL electrospinning.C figure shows that individual cells is at single PCL electricity Spin the growth conditions (arrow indication) on cotton fiber.In figure, cytoskeleton all dyes redness, and the nucleus of DAPI resisdye sends indigo plant Color fluorescence.PCL fiber is from fluoresced green.Scale: A, B=200 μm, C=100 μm.

Accompanying drawing 8: substantially see before and after acellular electrospinning film (cotton) scaffold for vascular tissue engineering subcutaneous transplantation.Intravascular stent is respectively It is paperwrapped in rubber tube is made (A and H) by tradition PCL electrospinning film (comparison) or large aperture PCL electrospinning cotton (present invention).Intravascular stent Make animal subcutaneous transplantation for building the engineering blood vessel with autologous tissue.The wherein closely electrospinning subcutaneous shifting of film intravascular stent Plant rear 4w (B, C and D) and 6w (E, F and G) to draw materials；Accordingly, 4w (I, J and K) after loose electrospinning cotton intravascular stent subcutaneous transplantation Draw materials with after 6w (L, M and N).Hurdle, the rightmost side one corresponding graft of display detach the cross section after rubber tube supporter (D, G, K and N)。

Accompanying drawing 9: acellular PCL electrospinning film (cotton) scaffold for vascular tissue engineering subcutaneous rat transplants tissue slice after 4w.Nothing Cell consolidation PCL electrospinning film and acellular large aperture PCL electrospinning cotton scaffold for vascular tissue engineering are transplanted to subcutaneous rat, take after 4w Material.The connective tissue parcel of the visible electrospinning film outer layer of electrospinning film (matched group) frozen section HE dyeing.Electrospinning intermembranous acellular and Extrtacellular matrix deposition (A and B).Masson dyeing has no blue collagen fiber (C).Cotton (present invention) paraffin section of electrospinning is shown in Cell is grown into electrospinning film holostrome, and extracellular matrix starts to deposit (D and E), the visible a small amount of blue collagen fiber of Masson dyeing (F).Scale: A, D=400 μm；B, C, E, F=100 μm.

Accompanying drawing 10: electrospinning film (cotton) subcutaneous rat is transplanted 6w and formed vascular tissue section.Acellular consolidation PCL electrospinning film and Acellular large aperture PCL electrospinning cotton scaffold for vascular tissue engineering is transplanted to subcutaneous rat, draws materials after 6w.Electrospinning film (matched group) ice Freezing section HE dyeing and see electrospinning film outer wrapping connective tissue, grow into without autogenous cell in electrospinning film, the most acellular epimatrix deposits (A and B).The outer minimal amount of collagen deposition of the connective tissue (C) of the Masson rarely seen electrospinning film of dyeing.Cotton (present invention) paraffin of electrospinning is cut The visible a large amount of cell of holostrome of sheet HE dyeing and extracellular matrix (D and E), Masson dyes and sees that blue Collagen fiber deposition increases, Extracellular matrix is hoop arrangement (F) of similar natural vessel wall.Scale: A, D=400 μm；B, C, E, F=100 μm.

Accompanying drawing 11: engineering blood vessel SMC markers immunofluorescence dyeing.With cotton (this of large aperture PCL electrospinning Invention) engineering blood vessel (subcutaneous rat 4w) the frozen section immunofluorescence dyeing that builds.Often row picture becomes one group, often in group Cytoskeleton is marked as red fluorescence (A, E and I), and in tissue, most cells SM22 label is positive (B), negative in tissue The new vessels tube wall of duty nutrition supply expresses SMA or Calponin (F and J).Nucleus DAPI resisdye be blue (C, G and K), often group picture stack result is respectively D, H and L.All picture scale=50 μm.

Detailed description of the invention

The detailed description of the invention provided the present invention below in conjunction with the accompanying drawings elaborates.

Embodiment 1

One, material and method

1 animal

150g male cleaning grade SD rat is the animal involved by this experiment, by the Shanghai limited public affairs of Si Laike laboratory animal Department provides.Animal protocols and experimental implementation are all tested by Shanghai Jiaotong University Medical College subsidiary Shanghai Children's Medi The examination and approval of the care of animal group.

2 instruments

3 reagent and material

4 methods

The preparation of 4.1 related solution

(1) 10% and 35% polycaprolactone (PCL) Static Spinning solution

With TFE as solvent, weigh PCL granule with electronic balance, turn after weighing in the solute ratio of 10% and 35% (m/v) Enter to dissolve bottle.The TFE measuring respective volume by the density electronic balance of 1.386g/ml proceeds to corresponding dissolving in bottle.Under room temperature Bottle promotion PCL uniform dissolution is dissolved in upset repeatedly.For Static Spinning after persistently 4d is dissolved in upset.The solution prepared should be in 1w It is finished.

(2) Smooth Muscle Cell liquid

Smooth Muscle Cell liquid is prepared in units of original-pack High-glucose DMEM culture fluid one bottle.With 500ml/ As a example by bottle High-glucose DMEM culture fluid, add 55ml hyclone (FBS) and 5.5ml penicillin streptomycin is dual anti- (PS).The High-glucoseDMEM Smooth Muscle Cell liquid containing 10%FBS and 1%PS it is made into after mixing.Culture fluid breaks a seal After being made into, it is placed in 4 DEG C of Refrigerator stores, should be finished in 2w.

The preparation of 4.2 Static Spinning PCL fibers

(1) preparation (present invention) that crude fibre PCL electrospinning is cotton

Draw 35%PCL Static Spinning solution with syringe, change 14G flat mouth dispensing needle head.Syringe is loaded into trace note Penetrate pump, and dispose the top being allowed to be suspended from hollow out four skeleton cylinder reception device.Regulation height, makes needle point and receives between device Distance be 6cm.High voltage power supply region end being connected to cylinder and receives device, power supply high-pressure side is sandwiched in syringe needle metal part.Adjust Joint high voltage power supply, making High voltage output is 16kV.Regulation micro-injection pump injection rate is 5ml/h.Regulation roller controller, makes rolling The cylinder speed of rotation is 2000rpm.At the Static Spinning initial stage, owing to wire vent is the most unstable, this part Electrospun can be collected in cylinder one end. After wire vent is stable, in the middle part of cylinder, receive Electrospun.Should the most slowly move left and right rolling during reception, make Electrospun exist With uniform Density Distribution on cylinder.

(2) preparation (matched group material) of tradition fine fibre PCL electrospinning film

Fine and closely woven PCL electrospinning mould is prepared: extract with syringe with reference to the method preparing nanofiber electrospinning film before us 10%PCL Static Spinning solution, uses 18G flat mouth dispensing needle head instead.The similar coarse-fibred preparation of PCL, is installed in the same way Micro-injection pump.With the solid roll that is coated with aluminium foil for receiving device.Receiving range between regulation syringe needle and cylinder is 12cm, the injection speed of micro-injection pump is 3ml/h, and high-voltage power voltage is 12kV, and drum rotation speed is 2000rpm.Collect Electrospinning film as the matched group of following experiments.

The morphologic observation of 4.3PCL electrospinning film (cotton)

(1) gross examination of skeletal muscle

Observe two kinds of different electrostatic spinning fibers form on collection device respectively, note fiber thickness, between fiber The indexs such as the depth of parallelism of arrangement.Multilamellar crude fibre PCL electrospinning cotton stacking can obtain " large aperture PCL electrospinning is cotton " and (be called for short " electrospinning Cotton ", i.e. the present invention)；The fiber that under peeling off, aluminium foil surface is assembled obtains " tradition PCL electrospinning film " (to be called for short " electrospinning film ", for this Bright matched group).Observe electrospinning film (cotton) form.

(2) sem observation and analysis

Two kinds of PCL electrospinning film (cotton) are placed in vacuum freezing drying oven and are dried overnight.Electrospinning film after preliminarily dried (cotton) processes through critical point drying, metalling again, is placed in scanning electron fibrescope observation.The picture ImageJ obtained 1.50i image analysis software processes, the angle of 150 fiber opposite planar coordinate x-axis and diameter in random measurement image, then unites The indexs (n=3) such as meter angular distribution, distribution of fiber diameters and average fibre diameter.The fibre diameter of bi-material is by average straight Footpath ± standard deviation represents, the statistical difference opposite sex uses independent samples t test (p < 0.05 is considered there is significant difference).

The mechanics detection of 4.4PCL electrospinning film (cotton)

PCL electrospinning film (cotton) fiber alignment received with cylinder has certain orientation characteristic, is therefore analyzing its mechanics During feature, point suitable machine direction mechanical characteristics and vertical fibers direction mechanical characteristics two aspect.By along machine direction and vertical fibre Dimension direction is rectangular length, and electrospinning film (cotton) is cut to the rectangular pieces of 10 × 40mm size, measures its thick (often group respectively Material n=5).During measurement, the upper and lower fixture of puller system is clamped rectangular pieces and is often held each 10mm, therefore includes the fibre plate of measuring in Length is respectively as follows: a=20mm, the thickness (mm) of b=10mm, c=vernier caliper measurement.After puller system starts, will measure Pulling force (f/kN) produced by (l/mm) material piece under different elongation length.

In drawing process, the pulling force (pressure) that material produces is calculated by following equation:

$Stress\left(mPa\right)=\frac{f\left(kN\right)/1000}{b\left(mm\right)\&CenterDot;c\left(mm\right)}$

In drawing process, the stretch ratio of the relatively primitive length of material is:

$Strain\left(\%\right)=\frac{\mathrm{\&Delta;}l\left(mm\right)}{a\left(mm\right)}\&times;100\%$

In drawing process, " pulling force-stretch ratio curve " is used for weighing the change that material is overall, curve linear ascent stage The Young's modulus that slope is material (Young ' s modulus), for weighing the ability (compliance) of material resistant deformation.Two The Young's modulus planting material is represented by " average diameter ± standard deviation ", and the significant difference of storeroom uses independent sample t inspection Test (p < 0.05 is considered there is significant difference).

The hydrophilic angle detection of 4.5PCL electrospinning film (cotton)

Hydrophilic angle is the detection material method to certain liquid affinity.The material hydrophilic angle i.e. material to water in this research With the contact angle (contactangle) of water, the latter refer to gas, liquid, solid three-phase cross place make liquid-vapor interface tangent line with Angle between solid-liquid boundary.By above-mentioned electrospinning film (cotton) after vacuum lyophilization by " fiber is parallel with photographic head " or " fiber is vertical with photographic head " two kinds of placement directions are respectively placed on the detector of hydrophilic angle, then drip pure water water droplet, record drop The hydrophilic angle (n=5) of 0s, 20s and 50s behind contact material surface.The hydrophilic angle of each time point after every time measuring is by " the most straight Footpath ± standard deviation " represent, the hydrophilic angle on every kind of material difference placement direction, the system at the hydrophilic angle of the identical placement direction of storeroom Meter is learned diversity and is used independent samples t test (p < 0.05 is considered there is significant difference).

The cytologic experiment of 4.6PCL electrospinning film (cotton)

(1) material prepares

In the Static Spinning film (cotton) of the suitable size of cutting is placed in 6 orifice plates respectively (often organizing n=3), each plate hole annular is not Material surrounding pushed down by rust steel, in order to avoid material migration in hole in operating process.The material 2h in hole is soaked with 75% ethanol for disinfection After, wash 3 times with sterile purified water.It is placed in lyophilizing in vacuum freezing drying oven.Irradiating surface sterilizing under uviol lamp before use 30～60min.

(2) cell prepares

Take P2～4 generation human artery's conduit smooth muscle cell.Suck the smooth muscle culture fluid in culture dish, with PBS rinsing 1～ After 2 times, add 1ml pancreatin/10cm culture dish.It is positioned in 37 DEG C of incubators digestion 2～3min.3 times of pancreatin are added after completing The Smooth Muscle Cell liquid of amount terminates digestion, moves into centrifuge tube.It is centrifuged 5min with the speed of 1000rpm.Siphon away supernatant, then A certain amount of Smooth Muscle Cell liquid is resuspended, and cell counting count board counts.

(3) plantation of cell

Equipped with 6 orifice plates of bi-material for Smooth Muscle Cell after (1) being processed.In advance often before cell inoculation Adding a certain amount of Smooth Muscle Cell liquid in hole, after making material fully absorb culture fluid, culture fluid can not have material completely. Smooth muscle cell after resuspended is pressed 5 × 10⁵The density in/hole is added drop-wise in each hole the most equably, and quiescent culture is in containing 5% CO₂37 DEG C of constant incubators.Every 2d changes liquid 1 time.

(4) Fluorescent Staining Observation of cell

Above-mentioned cell inoculated and cultured, after 6 days, siphons away culture fluid, washs 2～3 times with PBS, transfers to 6 new orifice plates, keep away Experimental result is interfered by the cell exempting from former 6 orifice plate bottom grown.30min is fixed with 4% paraformaldehyde.PBS rinsing 2～3 Secondary.Cytoskeleton fluorescence dye liquor by the proportions of 3.5:500 (v/v) and adds in plate hole with PBS, in room temperature dyeing 30min, PBS rinses 3 times.Adding the DAPI dye liquor resisdye nucleus 10s of 1:2000 dilution, tri-distilled water rinses 3 times.Fluorescence microscopy Observe.

5PCL electrospinning film (cotton) is for the structure of autologous tissue's engineered blood vessels

Building of 5.1 electrospinning film (cotton) scaffold for vascular tissue engineering

(1) the acellular PCL in large aperture electrospinning cotton scaffold for vascular tissue engineering build (organizational project blood of the present invention Pipe holder)

Crude fibre PCL electrospinning cotton is initially collected in hollow out four skeleton cylinder.With plantation rubber column jecket as inner support, by cylinder Crude fibre be paperwrapped in rubber tube surface 10 in the same direction and enclose, make large aperture acellular PCL electrospinning cotton engineering blood vessel Support.In order to avoid electrospinning cotton fiber scatters after winding, rubber tube terminal 7-0 silk thread is fixed.

(2) fine and closely woven acellular PCL electrospinning membrane tissue engineered blood vessels support build (comparison)

The building method of similar crude fibre scaffold for vascular tissue engineering.Fine and closely woven electrostatic spinning fiber film is peeled off from aluminium foil surface, It is wound in rubber cylinder tube-surface 10 again to enclose, makes fine and closely woven acellular PCL electrospinning membrane tissue engineered blood vessels support.Two ends after completing Fix with 7-0 silk thread.

The animal subcutaneous transplantation of 5.2 scaffold for vascular tissue engineering and evaluation

Should complete according to the method for narration in above-mentioned cytologic experiment before transplanting " 75% ethanol disinfection-distilled water flushing- Vacuum freeze-drying-ultraviolet sterilization " sterilization step.Meanwhile, also should soak with the normal saline containing antibiotic before transplantation Bubble.

With about 150g male SD rat for transplanting object.Experimental rat body weight is treated in weighing, with 0.3ml/100g body weight Dosage lumbar injection chloral hydrate anesthesia rat.After rat is lost consciousness, reject back hair with shaver.Use chelated iodine Skin in the range of sterile surgical position and surrounding 3～5cm.Single with sterile gauze paving.Cut the skin of suitable size, with hemostasis After pincers separate section skin and subcutaneous tissue, electrospinning cotton or electrospinning membrane tissue engineered blood vessels structural transplantation will be enclosed with to subcutaneous (often organizing n > 3).Notice that position, vascular stent graft place should be away from skin incision.With 4-0 band wire cutting needle after completing to transplant Skin suture.With chelated iodine sterilization wound 3 times.After operation completes at once, 1d and 2d intramuscular injection antibiotic prophylaxis infects.

The evaluation of 5.3 autologous tissue's engineered blood vessels

(1) gross examination of skeletal muscle

After scaffold for vascular tissue engineering subcutaneous rat is transplanted, 4w and 6w draws materials.Excess anesthesia is put to death the former operation of rat tailing edge and is cut Mouth cuts off skin.With mosquito forceps careful separation skin and subcutaneous tissue until engineering blood vessel manifests.Tissues observed engineering blood Tubing and the relation of surrounding tissue.Completely stripping engineering blood vessel, in extracting out, the rubber of parcel, notes tissue cross Face situation.Tissue elasticity is experienced with tweezers.

(2) paraffin section and dyeing

Routine paraffin wax is cut into slices and is dyeed, and completes tissue paraffin section de and HE dyeing, Masson trichrome stain.Microscope is observed Section position vascular tissue formational situation.Should especially notice whether tissue deep has cell to grow into.

(3) frozen section SMC markers immunofluorescence dyeing

Conventional frozen section and colouring method, with SM22, Calponin and SMA for SMC markers, and resisdye Cytoskeleton and nucleus, complete tissue freezing section and dyeing.Finally take pictures with fluorescence microscope.

Two, result

The preparation of 1PCL electrospinning film (cotton)

1ml concentration be 35% PCL solution can obtain arranging in flat by electrostatic spinning being collected on hollow out cylinder The crude fibre PCL electrospinning of row orientation is cotton.Optical microphotograph Microscopic observation fibres visible bending such as spring, major part fiber alignment orientation Unanimously.Fiber between hollow out skeleton is around between C-shaped iron wire two-arm in the same direction, available short texture after multiple-layer stacked Large aperture PCL electrospinning cotton (present invention).It is collected in solid rolling by electrostatic spinning with the PCL solution that 3.5ml concentration is 10% Available fine and closely woven fiber on cylinder.The fibrous membrane collected, i.e. " tradition PCL electrospinning film " (comparison) is taken off from aluminium foil surface, can Substantially experience the compact structure (Fig. 2) that fine and closely woven fiber accumulations becomes.

The sem observation of 2PCL electrospinning film (cotton)

Scanning electron microscope is to observe one of two kinds of electrospinning maximally effective instruments of film microstructure.In identical multiplying power condition Under, 10%PCL Static Spinning solution has the feature of traditional electrospinning film according to certain condition spun PCL electrospinning film: fiber is tiny (diameter about 0.67 ± 0.34 micron), and the hole formed between fiber is little；On the contrary, this research 35%PCL Static Spinning solution exists The PCL cellosilk received on hollow out cylinder is thick (diameter about 3.90 ± 1.85 μm), and the aperture that fiber architecture goes out is also the biggest In the former (about some tens of pm).Owing to using cylinder to collect, therefore two kinds of fiber alignment are also in obvious orientation characteristic (Fig. 3, figure 4)

The mechanical property of 3PCL electrospinning film (cotton)

Owing to the fiber of PCL electrospinning film (cotton) has certain orientations, therefore, the tensile property of material can be divided into suitable Machine direction and two kinds of vertical fibers direction situation.(1) along in machine direction, the pulling force suffered in unit are of material with Stretch ratio is the most linear, occurs a flex point afterwards, and unit are pulling force elongates with material and reduces.Fine In the moment of the PCL electrospinning film knee of curve respective material fracture of dimension consolidation, therefore the curve after flex point is down to " 0 " MPa suddenly；And PCL electrospinning that fiber is loose cotton after flex point its stress slowly reduce, actually appear that cotton piece fiber is single to rupture successively, and Non-integral ruptures.(2) on vertical fibers direction, bi-material equally exists the line of pulling force-elongation ratio within the specific limits Sexual relationship and flex point.But, the appearance position of flex point, relatively along lower in machine direction, from zero apart from farther, shows fibre Dimension vertical direction has higher compliance and less fracture stress.It should be noted that the tracing pattern of vertical direction in Zigzag.Young's modulus is to evaluate the physical quantity (compliance) of material opposing deformability, i.e. material pulling force-stretch ratio curve The slope of linear rise section.Though visible in suitable machine direction or vertical fibers direction, the Young that the PCL electrospinning of porous is cotton Modulus is all significantly less than PCL electrospinning film (Fig. 5) of consolidation.

The hydrophilic angle detection of 4PCL electrospinning film (cotton)

Hydrophilic angle may be used for the affinity weighing measured material with water, and affine angle is the least, and measured material is affine with water Property is the highest.Although PCL electrospinning film and the cotton hydrophobic material that belongs to of PCL electrospinning, but due to technology of preparing and material microstructure not With, between bi-material and the hydrophilic angle of the different directions of material own still differs.Consolidation PCL electrospinning film " fiber with take the photograph As head is disposed vertically direction " and the hydrophilic angle of " fiber with photographic head parallel direction " be all significantly greater than " the PCL electrospinning of porous Cotton ".For porous PCL electrospinning film, in " fiber and photographic head vertical direction ", its hydrophilic angle is significantly less than " fiber and photographic head Parallel direction ".At consolidation PCL electrospinning film, although the hydrophilic angle in " fiber and material vertical direction " has less than " fiber and material Parallel direction " trend at upper hydrophilic angle, but both contrasts do not have significant difference.Further, it is also possible to observe porous PCL electrospinning The trend (Fig. 6) that hydrophilic angle on film " fiber is vertical with photographic head " direction is the most constantly reduced.

The cytologic experiment of 5PCL electrospinning film (cotton)

After human smooth muscle cell is incubated at PCL electrospinning film or PCL electrospinning cotton 6d, cytoskeleton fluorescence staining observable Spread in fibrous membrane surface equably to the cell on PCL electrospinning film, but due to the planar structure of electrospinning film, cell does not has Three-dimensional space position difference.Cell on electrospinning film can arrange along fibre orientation, but the most obvious.Smooth muscle cell equally can be PCL electrospinning cotton grows.Owing to electrospinning cotton is loosened, and in distributed in three dimensions, therefore cell position is according to the spatial arrangements of fiber The most straggly.After Smooth Muscle Cells Adhesion is on electrospinning cotton fiber, cell space can stretch along fiber surface and extend (Fig. 7).

6PCL electrospinning film (cotton) is used for tissue engineering vessel

The large aperture PCL electrospinning bale of cotton is around to after rubber tube, it is seen that acellular electrospinning cotton intravascular stent surface is fluffy, and fine and closely woven PCL electrospinning film vascular stent material smooth surface is tight.Subcutaneous rat is drawn materials after transplanting 4w, it is seen that " electrospinning cotton intravascular stent group " Organize the most fully wrapped around electrospinning cotton support.Surface is one layer of loose connective tissue, tight through lower floor's flesh pink quality seen from top layer Close class vascular tissue.Rubber tube supporter can more easily with separate tissue.Engineering blood vessel cross section meat after separation It is cotton that eye is difficult to see original electrostatic spinning fiber, and tube chamber face is smooth.Drawing materials can for " the electrospinning film intravascular stent group " of transplanting 4w Seeing, surface has loose connective tissue to cover, and can see through lower floor's pale asphyxia " tissue ".But, during taking out rubber thing, The electrospinning film held separates, it is impossible to obtain complete engineering blood vessel." electrospinning cotton intravascular stent " is at subcutaneous transplantation 6w After result of drawing materials substantially similar with 4 weeks results of drawing materials from substantially seeing, surface has loose connective tissue to wrap up, and lower floor is fine and close meat Red engineering blood vessel.After electrospinning film intravascular stent group subcutaneous transplantation 6w, substantially see result of drawing materials with 4w similar, still cannot Take out complete vascular tissue (Fig. 8).

The histological observation of 7PCL electrospinning film (cotton) engineering blood vessel

The engineering blood vessel HE dyeing display formed after acellular electrospinning cotton intravascular stent subcutaneous transplantation 4w, cell is the longest Enter inside electrospinning cotton.Subregion cell has the feature of annular array.But, in histiocyte, cavity is more.Masson contaminates Color shows loose blue Collagen fiber deposition, and deposition site tissue is substantially fine and close than without deposition site.Electrospinning membrane support The blood vessel formed after transplanting is difficult to peel off due to support and cylindrical tube, therefore takes and separates the most representational outer tissue and make ice Freeze section.Visible this engineering blood vessel only outermost layer has loose connective tissue to wrap up, and internal layer electrospinning film is entirely without cell and thin Extracellular matrix, Masson dyeing shows collagen-free fiber deposition (Fig. 9) in tissue.

The engineering blood vessel HE section visible cell formed after the subcutaneous 6w of electrospinning cotton support has been fully grown in electrospinning cotton Side, blood vessel wall holostrome tissue tight, especially substantially increase during rubber standoff side cell and ECM density relatively 4w.Cell Epimatrix arranges ringwise, the basic arrangement conformation being similar to native blood vessels middle level.The dyeing of Masson trichrome stain visible blue Collagen fiber deposition substantially thickens and fine and close.Consolidation electrospinning membrane support group frozen section coloration result and 4w are without too big difference (figure 10)。

8 tissue smooth muscle markers tests

In order to detect whether the engineering blood vessel of subcutaneous formation has the cell that SMC markers is positive, experiment By tissue freezing section immunofluorescence dyeing SMC markers SM22, Calponin and SMA.Result shows, material After subcutaneous rat transplants 4w, there is a large amount of cell in the engineering blood vessel of structure, wherein most cells express smooth muscle cell Specific marker thing SM22.Calponin+ and SMA+ cell circular array, is the micro-blood being responsible for nutrition supply in blood vessel wall Tube wall dyeing (Figure 11).

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, on the premise of without departing from the inventive method, it is also possible to makes some improvement and supplements, and these improve and supplement and also should be regarded as Protection scope of the present invention.

Claims (10)

1. the electrospinning being used for preparing engineering blood vessel is cotton, it is characterised in that described electrospinning cotton is polycaprolactone electrospinning Cotton.

Electrospinning the most according to claim 1 is cotton, it is characterised in that described electrospinning cotton is large aperture parallel polycaprolactone electricity Spinning cotton, the aperture of electrospinning cotton is 20 μm～100 μm.

Electrospinning the most according to claim 1 is cotton, it is characterised in that described electrospinning cotton is the polycaprolactone of 30～40% The electrospinning that trifluoroethanol solution is obtained by electrostatic spinning is cotton.

Electrospinning the most according to claim 3 is cotton, it is characterised in that described electrospinning cotton prepares by the following method: configuration Mass volume ratio is the trifluoroethanol solution of the polycaprolactone of 35%, then carries out Static Spinning with the cylinder of hollow out for collection device Silk, it is thus achieved that electrospinning is cotton.

5. according to the cotton application at the support preparing engineering blood vessel of arbitrary described electrospinning of claim 1-4.

6. a scaffold for vascular tissue engineering, it is characterised in that described scaffold for vascular tissue engineering builds for utilizing electrospinning cotton Tubular bracket.

Scaffold for vascular tissue engineering the most according to claim 6, it is characterised in that described electrospinning cotton is that large aperture is parallel Polycaprolactone electrospinning is cotton.

Scaffold for vascular tissue engineering the most according to claim 6, it is characterised in that described scaffold for vascular tissue engineering Preparation method is: with cylinder as inner support, and large aperture polycaprolactone electrospinning cotton is paperwrapped in periphery in the same direction, Make scaffold for vascular tissue engineering.

Scaffold for vascular tissue engineering the most according to claim 8, it is characterised in that described scaffold for vascular tissue engineering Preparation method is: configuration quality volume ratio is the trifluoroethanol solution of the polycaprolactone of 35%, then with hollow out four skeleton cylinder Carry out electrostatic spinning for collection device and obtain electrospinning cotton, for inner support, electrospinning cotton is paperwrapped in the same direction with plantation rubber column jecket Rubber cylinder tube-surface, makes scaffold for vascular tissue engineering.

10. according to the application in preparing engineering blood vessel of the claim 6-9 arbitrary described scaffold for vascular tissue engineering.