CN105233345A - Natural protein/polycaprolactone nanofiber electrospun membrane, and preparation and application thereof - Google Patents
Natural protein/polycaprolactone nanofiber electrospun membrane, and preparation and application thereof Download PDFInfo
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Abstract
The invention provides a natural protein/polycaprolactone nanofiber electrospun membrane, and preparation and application thereof. The natural protein/polycaprolactone nanofiber electrospun membrane is obtained by mixing natural protein and polycaprolactone to prepare a spinning solution and performing electrospinning, and the natural protein comprises natural elastin and natural collagen. The natural protein containing cardiac muscle tissue extracellular matrix components is mixed with an artificially synthesized degradable material, and the nanofiber membrane can be prepared quickly and effectively through an electrostatic spinning technically. The natural protein/polycaprolactone nanofiber electrospun membrane has the advantages that preparation is simple, materials are easy to obtain, and preparation cost is low. The natural protein/polycaprolactone nanofiber electrospun membrane not only has a good biomechanical property, but also can fully simulate cardiac muscle tissue extracellular matrix environment. Biological simulation can be carried out in the aspects of the structure and the ingredient, and an effect of treating myocardial infarction by stem cell transplantation is improved. Therefore, the natural protein/polycaprolactone nanofiber electrospun membrane can serve as an extracellular culture medium to provide an extracellular matrix microenvironment for promoting cell adhesion, survival and multiplication.
Description
Technical field
The present invention relates to the preparation containing native protein/PCL nanofiber composition electrospinning film and the basic research in cell culture and organizational project thereof and application, belong to biological medicine, tissue engineering, cytobiology, stem cell biology, biomaterial and cardiovascular injuries surgical field.
Background technology
Ischemic heart desease, as one of China's major health concern, brings heavy financial burden to China and other countries of the world.Although current medicine, intervention and surgical operation therapy ischemic heart desease effectively can improve blood supply of cardiac muscle, save dying cardiac muscle, but for murderous nonfunctional cardiac muscle not repair.Serious arrhythmia, heart failure and sudden cardiac arrest that large-area Myocardial death can cause.For the treatment of advanced heart disease, comparatively effective method is heart transplantation at present.But the donor amount of heart transplantation is few, the factors such as strong immunological rejection add its medical treatment cost and cause it to be difficult to widespread adoption clinically.
Myocardial cell is the material base having participated in heart series of complex function as the elementary cell that heart forms.Existing research is reported, is applied to the method that somatic stem cell transplants and can treats mice ischemic heart desease, comprise and improve ejection fraction after heart infarction, stop left ventricular remodeling, and increase number of myocardial cells by the differentiation of stem cell.Therefore stem cell transplantation technology receives increasing concern, for treatment ischemic heart desease provides a kind for the treatment of prospect completely newly.
Stem cell classification is various, wide material sources.Bone marrow stem cell is existing in Myocardial Regeneration and heart and injury reparation to be reported widely.Wherein, c-kit+ Bone Marrow Stem Cells Transplantation enters in Mice Body can reduce mice myocardial infarct size, improves left ventricular ejection fraction, and have also appeared newborn cardiac muscle in mice heart infarction surrounding zone.These research appropriate transplanting modes of prompting and correct stem cell select the revascularization that can promote the regeneration of infarcted region myocardial cell and dying district cardiac muscle.
Intravascular injection and myocardium in situ injection are the main method that damaged myocardium is repaired in current stem cell transplantation.Intravascular injection is that a kind of wound is little, Therapeutic Method simple to operate, but after intravascular injection stem cell, the quantity of stem cell homing, the factors such as immunological rejection constrain its curative effect.Although and myocardium in situ injection method can ensure some stem cell exist in the local of damaged myocardium, due to its injury of myocardium normal organization and the factors such as injection site limitation greatly limit its clinical practice widely.And myocardial cell is present in a large amount of epimatrix (ECM); its main component is collagen protein and the elastic fiber of fibroblast generation and secretion in myocardium gap; these extracellular matrixs play an important role to the normal morphology and function of maintenance heart; myocardial collagen fiber is interweaved; form multi-level, multi-faceted, complicated three-D space structure, play a significant role in support, protecting myocardial cell, coordination myocardial contraction, diastole etc.Therefore, the environment setting up a kind of similar myocardium substrate for bone marrow stem cell is by advantageously in its reparation to myocardial defect.
In current research, polycaprolactone (Polycaprolactone, PCL) as the linear aliphatic adoption ester obtained by 6-caprolactone ring-opening polymerisation, there is good heat stability, biological degradability, hemicrystalline, mechanical property, drug permeability and biocompatibility, be often used to cardiac muscle tissue engineering.By stem cell transplantation, form " timbering material-stem cell " complex, treatment myocardial infarction.But due to the sheet mechanism that cardiac muscle is a kind of Nano grade, traditional support cannot accurate simulation cardiac muscle matrix environment.Therefore, the present invention by electrostatic spinning technique simple and quick prepare nanofiber, utilize its ultrastructure not only can simulate extracellular Matrix environment, promote that cell grows in fibrous membrane, and larger specific surface area strengthens intercellular contacting with each other, and is beneficial to the interaction between cell.
The topmost formation component of cardiac bistiocyte's epimatrix and collagen protein and elastin.Therefore the present invention has founded application electrostatic spinning technique and native protein (collagen protein/elastin) and polyester material polycaprolactone (PCL) has been made nano fibrous membrane, the hydrophobicity of native protein to aliphatic polyester series material polycaprolactone (PCL) is utilized to carry out modification, reinforced polymeric material to the adhesion of cell, thus simulates extracellular Matrix environment more fully.This nanofiber has good biocompatibility and mechanical property, can improve the therapeutic effect of Effects of Stem Cell Transplantation on Myocardial Infarction Patients from structure and bionical two aspects of composition, thus realizes the Biofunctional of " timbering material-stem cell " complex.Therefore, the present invention proposes: native protein (collagen protein/elastin) and polycaprolactone (PCL) are made electrospinning film, compound bone marrow C-kit
+complex-myocardium composite patch that stem cell kind is made " timbering material-stem cell ", repairs damaged myocardium, improves cardiac function after heart infarction.
Summary of the invention
The object of this invention is to provide a kind of native protein component compound PCL nanofiber electrospinning film of simulating extracellular Matrix environment also prepare myocardium sticking patch by plantation stem cell thus repair damaged myocardium, reach the object for the treatment of myocardial infarction.
In order to achieve the above object, the invention provides a kind of native protein/polycaprolactone nanofiber electrospinning film, by by native protein and polycaprolactone mixed preparing spinning liquid, form through electrospinning, described native protein comprises natural resiliency albumen (elastin) and natural collagen protein (collagen).
Present invention also offers the preparation method of above-mentioned native protein/polycaprolactone nanofiber electrospinning film, it is characterized in that, comprise: natural resiliency albumen and natural collagen protein are mixed into native protein mixture according to mass ratio 1: 9 ~ 3: 7, described native protein mixture is mixed according to mass ratio 2: 8 ~ 8: 2 with polycaprolactone, be dissolved in hexafluoroisopropanol or trifluoroethanol, stir, obtain spinning liquid, electrospinning, described electrospinning condition is: voltage is 15kV, receiving range is 12.5cm, injection rate is 1.2ml/h, ambient temperature is room temperature, relative humidity is 20-80%, obtain native protein/polycaprolactone nanofiber electrospinning film.
Preferably, described stirring is for adopting magnetic stirrer 24h.
Preferably, described native protein mixture and the mass volume ratio of hexafluoroisopropanol or trifluoroethanol are 15% (0.15g/ml) (in g/ml).
Preferably, gained native protein/polycaprolactone nanofiber electrospinning film drying, sterilize for subsequent use.
Present invention also offers above-mentioned native protein/polycaprolactone nanofiber electrospinning film as the application of tissue engineering bracket in preparation tissue-engineering graft constructed.
Preferably, the tissue-engineering graft constructed that described tissue-engineering graft constructed is for repairing ischemia myocardial damage, improve cardiac function, improve the myocardial remodelling that heart failure or other reasons cause.
Preferably, described tissue-engineering graft constructed is the tissue-engineering graft constructed for repairing other tissue defect diseases except heart defect.
Present invention also offers above-mentioned native protein/polycaprolactone nanofiber electrospinning film as the application in cell injuring model medium.Described cell injuring model medium can be used for providing and promotes cell adhesion, survival, the microenvironment of propagation and simulation extracellular Matrix framework.
Present invention also offers a kind of cardiac patch, it is characterized in that, comprise seed cell and above-mentioned native protein/polycaprolactone nanofiber electrospinning film.
Preferably, described seed cell is the c-kit of derived from bone marrow
+stem cell, BMNC, mescenchymal stem cell, cardiac muscle dedifferente at least one in the cell of cell, Cardiac Stem Cells and derived from embryonic stem cells.
Preferably, described bone marrow C-kit
+stem cell is the C-kit of bone marrow derived
+stem cell, its preparation method comprises: the femur of the mice of stalk after 3 days of coring and tibia, go out bone marrow with Hanks balanced salt solution, isolate BMNC, magnetic bead sorting obtains c-kit
+stem cell.
Present invention also offers the preparation method of above-mentioned cardiac patch, it is characterized in that, comprising: by seed cell kind on above-mentioned native protein/polycaprolactone nanofiber electrospinning film, cultivate and obtain myocardium sticking patch.
The present invention demonstrate this cardiac patch to the reparation situation of cardiac function and containing the electrospinning film of native protein and degradable macromolecule material as simulation extracellular Matrix environment for Stem cells cultured in vitro provides promotion cell adhesion, survival, the place of propagation and extracellular matrix skeleton.
Compared with prior art, the invention has the beneficial effects as follows:
1. the present invention is by the fallen material mixing of the native protein containing cardiac bistiocyte's epimatrix composition and synthetic, and quick by electrostatic spinning technique, effectively make nano fibrous membrane, have simple to operate, material easily obtains, the advantage that cost of manufacture is cheap.Guarantee commercial possibility from now on.Utilize the respective advantage of native protein and synthetic degradation material, not only retain good biomechanical property, and fully simulate extracellular Matrix environment.Carry out human simulation from structure and composition two aspect, improve the therapeutic effect of Effects of Stem Cell Transplantation on Myocardial Infarction Patients.Therefore promotion cell adhesion can be provided as extracellular culture medium, survival, the extracellular matrix microenvironment of propagation.
2. existing bibliographical information, bone marrow C-kit
+stem cell can cells into cardiomyocytes, vascular smooth muscle, and endotheliocyte direction breaks up, and plays therapeutical effect in ischemic heart desease.But report bone marrow C-kit
+stem cell in vitro multiplication capacity is poor, and the time-to-live is short.Electrostatic spinning containing native protein and synthetic degradation material in the present invention can promote bone marrow C-kit in vitro
+the a large amount of increasings increment of stem cell, thus manufacture the seed cell of abundant quantity for cell transplantation.The present invention proposes first by bone marrow C-kit
+stem cell is planted and make cardiac patch on the electrospinning film containing native protein composition and degradable substance mixing.
3. in the present invention, also find that the electrospinning film containing native protein composition and degradable substance mixing has the effect promoting cell adhesion, propagation in vitro, be transplanted to heart infarction myocardial surface and effectively can improve cardiac function after heart infarction, organize left ventricular remodeling, play the stem-cell therapy effect of ischemic heart desease.
Accompanying drawing explanation
Fig. 1 is the electromicroscopic photograph of matched group and native protein/polycaprolactone nanofiber electrospinning film; (amplification 1.5k doubly)
Fig. 2 A is the contact angle picture of each native protein/polycaprolactone nanofiber electrospinning film; Fig. 2 B is each native protein/polycaprolactone nanofiber electrospinning film contact angle cartogram; #, &, * representative and 20% native protein (NP) group, the electrospinning film that in 50% native protein (NP) group, identical native protein proportioning is formed and matched group compare P < 0.05.
Fig. 3 is biomechanical characterization when native protein ratio elastin is 3: 7 with collagen protein quality ratio in each component electrospinning film; A: the Young's modulus of each group electrospinning film (Young ' smodulus); B: the percentage elongation (elongation) of each group electrospinning film; C: the fracture strength (tensilestrength) of each group electrospinning film;
Fig. 4 schemes for adhering to containing native protein and synthetic degradation material electrospinning film and bone marrow C-Kit+ stem cell; Each component electrospinning film and seed cell adhere to upper in figure row as the cultivation light microscopic photo of the 0th day, and lower row is the cultivation light microscopic photo of 10 days; 50 μm, scale.
Fig. 5 A, B, C are that each component electrospinning film body promotes bone marrow C-kit+ stem cells hyperplasia figure outward; Immunofluorescence dyeing proliferation marker Ki67 is added, the shows fluorescent microscopy images after ph3 also dyes after A: bone marrow C-kit+ stem cell cultivates 5 days; B: bone marrow C-kit+ stem cell is at each component electrospinning film and the direct growth curve cultivated in culture plate; C: be the proliferative conditions of B figure the 5th day time point; 50 μm, scale.#, &, * represent each component and compare with Cellsonly and purePCL group, P < 0.05.
Fig. 6 A is photo before each component electrospinning film is transplanted, and area is 0.5 × 0.5cm, thickness about 50 μm; B is the picture that electrospinning film is transplanted to mouse heart surface; C is transplant operation electrospinning film photo on mouse heart after 28 days, and on electrospinning film, visible new vessels is formed;
Fig. 7 A is that the cardiac patch made based on above-mentioned electrospinning film transplants latter 1 week, the normal group of 4 weeks, the ultrasonic figure of heart infarction non-treatment group typical heart; B is EF (EjectionFractions, the ejection fraction) cartogram of transplanting latter 7 days ultrasound detection, and C is FS (Fractionalshortening, the ventricle LVFS) cartogram of transplanting latter 7 days ultrasound detection; D is the EF cartogram of transplanting latter 28 days ultrasound detection; E is the FS cartogram of transplanting latter 28 days ultrasound detection; * represent cardiac patch group (80NP20PCL+cells with purePCL+cells) and compare P < 0.05 with the non-treatment group of heart infarction (MI) and simple electrospinning film acellular group (80NP20PCL with PurePCL), # represents cardiac patch group (80NP20PCL+cells) and the non-treatment group MI of heart infarction, non-repopulating cell group 80NP20PCL, PurePCL and purePCL+cells group compares P < 0.05.
Fig. 8 be heart infarction draw materials after 28 days obtain mouse heart TTC dye, the difference of myocardial infarct size between visible each experimental group.White portion is murine myocardial infarction region.Cardiac patch treatment group (80NP20PCL+cells) myocardial infarct size is less than MI group, purePCL+cells group and non-repopulating cell group.This result supports the result of cardiac ultrasonic, and namely 80NP20PCL+cells group mice myocardial infarct size is minimum, and cardiac function recovers best.
Detailed description of the invention
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
Application builds myocardium surgical repair infarcted myocardium containing native protein component and synthetic degradation material electrospinning film:
1. native protein/polycaprolactone nanofiber electrospinning film and structure thereof:
A kind of native protein/polycaprolactone nanofiber electrospinning film, by by native protein and polycaprolactone mixed preparing spinning liquid, form through electrospinning, described native protein is the combination of natural resiliency albumen and natural collagen protein.The preparation method of described native protein/polycaprolactone nanofiber electrospinning film is: by natural resiliency albumen (Elastin) (the Shanghai chemical Science and Technology Ltd. of profit meaning, 9007-58-3, China) and natural collagen protein (CollagenI) (sigma, C9879, the U.S.) be mixed into 1: 9 in mass ratio, the native protein mixture of 2: 8,3: 7.By native protein mixture and Biodegradable polyester class material polycaprolactone (PCL, (sigma, 440744-5G, the U.S., molecular weight 80000) mixing.The mass ratio of native protein mixture and PCL is 2: 8 (in corresponding diagram 1 20NP/80PCL), 5: 5 (in corresponding diagram 1 50NP/50PCL), 8: 2 (in corresponding diagram 1 80NP/20PCL).
Be dissolved in hexafluoroisopropanol by the mixture of gained, described native protein mixture and the mass volume ratio 15% of hexafluoroisopropanol, magnetic stirring apparatus stirs 12h.The spinning liquid prepared is proceeded in 10ml syringe, under the voltage of 15kV, control receiving range in 12.5 centimeters, electrospinning is carried out with the injection rate of 1.2ml/h, ambient temperature is room temperature, relative humidity is (20-80) %, electrospinning device purchased from Beijing Kai Weixin Science and Technology Ltd., drying, sterilize for subsequent use.As a control group, control identical spinning condition, prepare pure PCL nano fibrous membrane.Its thickness is the om observation for cell culture of 5 μm, thickness be 50 μm for transplantation treatment myocardial infarction after cell culture.Its electromicroscopic photograph as shown in Figure 1.
Application sessile drop method measures electrospinning film contact angle: the distilled water of at least 8 0.25 μ l drips to the diverse location of material at random.Material surface obtains drop shape photo after there is not obviously change, and analyzes the angle of water droplet and plane.As can see from Figure 2 elastin composition and electrospinning film hydrophilic proportional; Total protein content and hydrophilic are inverse ratio; 80%NP component electrospinning film has good hydrophilic, has the bionical feature of good biological, can as the rear benefit sticking patch of the cardiac patch for the treatment of heart infarction.
The material detector that 10 newton's loads are born in application detects electrospinning film mechanics feature (H5K-S, Hounsfield, UK), detect specimen and require rectangle (30 × 10 × 0.10-0.15mm), speed 10mm/min, often kind of component electrospinning film at least detects 5 times.(Figure 3 shows that each component electrospinning film mechanics feature.A: each component electrospinning film Young's modulus (Young ' smodulus); B: each component electrospinning film percentage elongation (elongation); C: each component electrospinning film fracture strength (tensilestrength); Although 80%NP component electrospinning film does not have best mechanics feature, according to operation technique requirement, its mechanical property is enough to reach the follow-up mechanics standard as sticking patch.
2. be separated c-kit positive cell:
Get 6-8 week age C57BL/6 mice ligation ramus descendens anterior arteriae coronariae sinistrae prepare mice heart infarction model.Core the mice of stalk after 3 days, de-neck puts to death mice, is separated femur, tibia, goes out bone marrow with the Hanks balanced salt solution (Biowest, L0606, France) containing 10%FBS.In 15ml centrifuge tube, add 3ml lymphocyte separation medium (reaching section is, DKW33-R0100, China), tile bone marrow suspension separating medium ullage, keeps two liquid level interfaces clear.Room temperature, adopts horizontal rotor to be (400-500rpm/min) centrifugal 20 minutes under the condition of 400g at centrifugal force.After centrifugal, be red blood cell layer at the bottom of pipe, intermediate layer is separating medium, and the superiors are blood plasma/tissue homogenate layers, and plasma layer is the finer and close tunica albuginea of skim, i.e. BMNC layer with being separated between liquid layer.Careful absorption tunica albuginea layer is in another centrifuge tube.Add equivalent PBS dilution, suction pipe piping and druming mixing.Room temperature, adopts horizontal rotor centrifugal 5min under the condition of 1500rpm/min, abandons supernatant.Add 3mlstainingbuffer (mixing of 1%BSA and 0.02%EDTA (sigma, RNBD0725, the U.S.) equal-volume is obtained) resuspended, room temperature, adopts horizontal rotor centrifugal 5min under the condition of 1500rpm/min, abandons supernatant.(its compound method is: get 1gBSA (sigma, SLBJ9162ZV, the U.S.) and be dissolved in 99g distilled water to add 3ml1%BSA, 0.22 μm of strainer filtering) resuspended closed, room temperature, adopts horizontal rotor centrifugal 5min under the condition of 1500rpm/min, abandons supernatant.The stainingbuffer adding 80 μ l is resuspended, adds the anti-mouse-c-kit magnetic bead (U.S. sky Ni) of 20 μ l after cell suspension, piping and druming mixing, 4 DEG C, rotates lucifuge and hatches, 15 minutes.After hatching, add 400 μ lStainingbuffer, final volume is 500 μ l; Detached dowel is placed in magnetic field, makes it flow down with 0.5mlStainingbuffer, detached dowel balances 3 times; Cross post and collect negative cells: cell suspension is loaded on the detached dowel after balance, carried out post; Collect c-kit negative cells; Repeat twice with the Stainingbuffer of 0.5ml; Collect the negative cells suspension of 1.5ml.(in triplicate, 2ml suspension can be collected according to cell concentration).Collect positive cell: after detached dowel and Magnet being separated, add 1mlstainingbuffer, and avoid producing bubble by lower plunger gently, collect c-kit positive cell (being bone marrow C-kit+ stem cell); (also naturally flow down can).By the cell mixing of collecting, counting chamber counts.
3. cardiac patch and preparation method thereof:
A kind of cardiac patch, comprises seed cell and above-mentioned elastin is the native protein/polycaprolactone nanofiber electrospinning film of 3: 7 with collagen protein quality ratio.Its preparation method is: step 2 is separated the bone marrow C-kit+ stem cell that obtains with 1 × 10
5individual/cm
2density kind on the native protein described in step 1/polycaprolactone nanofiber electrospinning film.With the FBS (Biowest containing 20%, S1810, France) IMDM culture medium (Lot.12440-053, Gibco, the U.S.) cultivate 20 days in 37 DEG C of incubators of 5%CO2, half amount changes liquid every three days, obtains cardiac patch (80NP20PCL+cells, 50NP50PCL+cells and 20NP80PCL+cells).As shown in Figure 4, cell and acellular matrix adhere to well.
Step 2 being separated the bone marrow C-kit+ stem cell obtained is inoculated on pure polycaprolactone nanofiber electrospinning film with equal densities, adopts the same terms to cultivate, obtains cardiac patch (purePCL+cells).
4. detect cardiac valve acellular matrix to the impact of cultured cell in vitro:
Get step 2 and be separated the bone marrow C-kit+ stem cell obtained, with density 3 × 10
5individual/cm
2plant on the native protein/polycaprolactone nanofiber electrospinning film (elastin is 3: 7 with collagen protein quality ratio) described in step 1 of 0.5 × 0.5cm size, be placed in culture plate and cultivate.The bone marrow C-kit+ stem cell of getting equivalent is directly placed in culture plate and cultivates, culture medium and condition of culture identical with step 3.Cultivation the 5th day, get the bone marrow C-kit+ stem cell (have and do not have acellular matrix) of cultivation, paraformaldehyde fixed 15 minutes, 0.5%Triton permeable membrane 15 minutes, and PBS micro oscillation washs 3 times, 5 minutes/time.5% donkey serum (Jakson, 109558, the U.S.) room temperature closes 1 hour; Add the mixed liquor indicating ki67 (BD, 556003, the U.S., thinner ratio 1: 200) and ph3 (cellsignaling, 9701S, the U.S., thinner ratio 1: 300) by the immunofluorescence dyeing propagation of 1%BSA dilution of 100 μ l; 4 DEG C of refrigerator overnight incubation; Hatch rear PBS micro oscillation and wash 3 times, 5 minutes/time; The PBS adding 100 μ l dilutes two anti-donkeyanti-mouseAlexa555 (Invitrogen, 1117032, USA) and ldonkeyanti-rabbitAlexa488 (Invitrogen, 1531671, USA) mixed liquor, dilution ratio is 1: 500; Incubated at room 1 hour.Hatch rear PBS micro oscillation and wash 3 times, 5 minutes/time; DAPI dyes 30 seconds, and PBS washes twice, 5 minutes/time.Fluorescence microscope.Result as shown in Figure 5A, compared with cultivating with simple cell, breed active on electrospinning film by cell.
Same, get step 2 and be separated the bone marrow C-kit+ stem cell obtained, with density 3 × 10
5individual/cm
2plant on the electrospinning film containing native protein, be placed in culture plate and cultivate.The bone marrow C-kit+ stem cell of getting equivalent is directly placed in culture plate and cultivates, culture medium and condition of culture identical with step 3.Added MTT (promega, G5421, the U.S.) at the 0th, 1,3,5,7,10,15 day respectively by 20 μ l/100 μ l culture medium, hatch 4 hours at 37 DEG C of incubators of 5%CO2, monitoring absorption photometric value.As shown in Fig. 5 B, C, MTT growth curve result shows that high protein content component can promote cell proliferation.
5. myocardium sticking patch is to the reparation situation of cardiac function:
Get the C57BL/6 mice in 6 week age, isoflurane inhalation anesthesia, be fixed on by the mice of having anaesthetized on Mus plate, preserved skin, cuts off skin of chest, at incision purse string suture, stays line to do for subsequent use.Elastic separating plier separating muscle, little curved forceps punctures into thoracic cavity along the 4th intercostal, retracts lower edge rib, extrudes heart, and 7-0prolene toe-in is pricked ramus descendens anterior arteriae coronariae sinistrae and made heart infarction model.
By 80%NP/PCL electrospinning film (80NP20PCL, elastin is 3: 7 with collagen protein quality ratio) obtained in cardiac patch (80NP20PCL+cells and purePCL+cells) obtained for step 3, step 1 with do not plant bone marrow C-kit
+the simple PCL electrospinning film (PurePCL) of stem cell is transplanted to heart infarction region respectively, (as Fig. 6 A: for transplanting front each component electrospinning film photo; B: cardiac patch has been transplanted on heart) heart also to be received, aerofluxus, closes thoracic cavity, will reserve siding knotting.Transplant 1 week, after 4 weeks, row toy cardiac ultrasonic monitoring mouse core function status.By normal mouse as a control group (control), mice (MI) row toy cardiac ultrasonic is not treated to heart infarction simultaneously.
As shown in Figure 7 A: the cardiac ultrasonic typical picture of transplanting 7 days and 28 days; 7B: pure heart infarction group, its poor heart function, heart infarction after 7 days ejection fraction (EF) value be 25.9 ± 2.5, transplant plantation bone marrow C-kit+ stem cell cardiac patch group after 7 days EF value be respectively 45.4 ± 2.5 (80NP20PCL+cells groups) and 43.0 ± 2.4 (purePCL+cells groups), transplanting do not plant bone marrow C-kit+ stem cell cardiac patch group after 7 days EF value be respectively 35.1 ± 3.4 (80NP20PCL groups) and 32.2 ± 1.6 (purePCL groups).7C:pureMI group, heart infarction after 7 days ventricle LVFS (FS) value be 12.7 ± 1.5, transplant plantation bone marrow C-kit+ stem cell cardiac patch group after 7 days EF value be respectively 23.8 ± 2 (80NP20PCL+cells groups) and 21.8 ± 1.1 (purePCL+cells groups), transplanting do not plant bone marrow C-kit+ stem cell cardiac patch group after 7 days EF value be respectively 17.4 ± 1.8 (80NP20PCL groups) and 16.1 ± 0.9 (purePCL groups).
Fig. 7 D: pure heart infarction non-heart transplant sticking patch group, its heart infarction after 28 days EF value be 38.9 ± 3.7, transplant plantation bone marrow C-kit+ stem cell cardiac patch group after 28 days EF value be respectively 55.8 ± 2.8 (80NP20PCL+cells groups) and 47.2 ± 1.6 (pure PCL+cells group), transplanting is not planted bone marrow C-kit+ stem cell cardiac patch group EF value and is respectively 46.3 ± 4.6 (80NP20PCL groups) and 45.7 ± 5.1 (purePCL groups).7E:pureMI group, heart infarction after 28 days ventricle LVFS (FS) value be 18.6 ± 1.9, transplant plantation bone marrow C-kit+ stem cell cardiac patch group after 28 days ventricle LVFS (FS) value be respectively 28.2 ± 2 (80NP20PCL+cells groups) and 23.8 ± 1.0 (purePCL+cells groups), transplanting do not plant bone marrow C-kit+ stem cell cardiac patch group after 7 days EF value be respectively 22.7 ± 1.8 (80NP20PCL groups) and 22.2 ± 2.1 (purePCL groups).
Transplant the latter 28 days all de-necks of all mices to put to death.When putting to death mice, visible heart surface is with complete cardiac patch, and on sticking patch, visible more rich vascular plexus is newborn.(Fig. 6 C: to transplant after heart infarction 28 days, the visible complete heart sticking patch of heart surface.Its visible new vessels in surface is formed) after PBS rinses, carry out TTC dyeing, white portion is heart infarction position, as shown in Figure 8.Cardiac patch treatment group (comprising repopulating cell and non-repopulating cell group) myocardial infarct size is less than pure MI group.In cardiac patch treatment group, 80NP20PCL+cells group, myocardial infarct size is minimum.
Claims (10)
1. native protein/polycaprolactone nanofiber electrospinning film, by by native protein and polycaprolactone mixed preparing spinning liquid, form through electrospinning, described native protein comprises natural resiliency albumen and natural collagen protein.
2. the preparation method of native protein according to claim 1/polycaprolactone nanofiber electrospinning film, it is characterized in that, comprise: natural resiliency albumen and natural collagen protein are mixed into native protein mixture according to mass ratio 1: 9 ~ 3: 7, described native protein mixture is mixed according to mass ratio 2: 8 ~ 8: 2 with polycaprolactone, be dissolved in hexafluoroisopropanol or trifluoroethanol, stir, obtain spinning liquid, electrospinning, described electrospinning condition is: voltage is 12-18kV, receiving range is 10-20cm, injection rate is 1.2ml/h, ambient temperature is room temperature, relative humidity is 20-80%, obtain native protein/polycaprolactone nanofiber electrospinning film.
3. the preparation method of native protein/polycaprolactone nanofiber electrospinning film as claimed in claim 2, it is characterized in that, the mass volume ratio of described polycaprolactone/native protein mixture and hexafluoroisopropanol or trifluoroethanol is 15%.
4. native protein according to claim 1/polycaprolactone nanofiber electrospinning film is as the application of tissue engineering bracket in preparation tissue-engineering graft constructed.
5. native protein/polycaprolactone nanofiber electrospinning film as claimed in claim 4 is as the application of tissue engineering bracket in preparation tissue-engineering graft constructed, is characterized in that, described tissue-engineering graft constructed is for repairing ischemia myocardial damage, improve cardiac function, improve the tissue-engineering graft constructed of the myocardial remodelling that heart failure or other reasons cause.
6. native protein/polycaprolactone nanofiber electrospinning film as claimed in claim 4 is as the application of tissue engineering bracket in preparation tissue-engineering graft constructed, it is characterized in that, described tissue-engineering graft constructed is the tissue-engineering graft constructed for repairing other tissue defect diseases except heart defect.
7. native protein according to claim 1/polycaprolactone nanofiber electrospinning film is as the application in cell injuring model medium.
8. a cardiac patch, is characterized in that, comprises seed cell and native protein according to claim 1/polycaprolactone nanofiber electrospinning film.
9. cardiac patch as claimed in claim 8, it is characterized in that, described seed cell is the c-kit of derived from bone marrow
+stem cell, BMNC, mescenchymal stem cell, cardiac muscle dedifferente at least one in the cell of cell, Cardiac Stem Cells and derived from embryonic stem cells.
10. the preparation method of cardiac patch according to claim 8, is characterized in that, comprising: by seed cell kind on above-mentioned native protein/polycaprolactone nanofiber electrospinning film, cultivates and obtains myocardium sticking patch.
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| CN107519524A (en) * | 2017-09-21 | 2017-12-29 | 滨州医学院 | A kind of polycaprolactone/collagen/quaternary ammonium salt electrospun composite fibers film and preparation method thereof |
| CN108079378A (en) * | 2017-12-22 | 2018-05-29 | 中国科学院上海硅酸盐研究所 | Compound myocardium patching material of the organic/inorganic of active plasma diffusing W,Mo function and nanostructured and preparation method thereof |
| CN109330740A (en) * | 2018-09-18 | 2019-02-15 | 武汉纺织大学 | A method of reducing artificial blood vessel permeance property |
| CN110404117A (en) * | 2018-04-28 | 2019-11-05 | 国家纳米科学中心 | A kind of functionalization guidance muscular tissue repair membrane and its preparation method and application |
| CN112851923A (en) * | 2019-11-12 | 2021-05-28 | 上海竞微扶生医学科技有限公司 | Modified polycaprolactone implant material and preparation method thereof, fiber and preparation method thereof, and patch |
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| CN114870089A (en) * | 2022-01-17 | 2022-08-09 | 中国人民解放军联勤保障部队第九八八医院 | PCL-collagen/Gelma biradial composite stent loaded with MSCs and application |
| CN115068687A (en) * | 2022-07-08 | 2022-09-20 | 重庆科技学院 | Gradient nano/microfiber scaffold and preparation method and application thereof |
| CN115068687B (en) * | 2022-07-08 | 2023-12-12 | 重庆科技学院 | Gradient nano/micro fiber support and preparation method and application thereof |
| CN115804869A (en) * | 2022-10-26 | 2023-03-17 | 王丽 | BADSCs membrane and conductive nanofiber composite heart patch and preparation method thereof |
| CN115804869B (en) * | 2022-10-26 | 2024-07-23 | 王丽 | BADSCs diaphragm and conductive nanofiber composite heart patch and preparation method thereof |
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