CN103893819A - Coaxial electrostatic spinning fibrous scaffold and preparation method thereof - Google Patents
Coaxial electrostatic spinning fibrous scaffold and preparation method thereof Download PDFInfo
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- CN103893819A CN103893819A CN201410105964.2A CN201410105964A CN103893819A CN 103893819 A CN103893819 A CN 103893819A CN 201410105964 A CN201410105964 A CN 201410105964A CN 103893819 A CN103893819 A CN 103893819A
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Abstract
The invention discloses a coaxial electrostatic spinning fibrous scaffold, belonging to the field of biological tissue engineering. The scaffold comprises an aliphatic polyester shell coupled with bone marrow derived mesenchymal stem cell (BMSC) affinity peptides and a polyvinylpyrrolidone core loading a recombinant human transforming growth factor (TGF)-beta1, not only is safe and non-toxic and has excellent biocompatibility but also can collect more BMSCs and promote chondrogenic differentiation of BMSCs, and then more cartilage tissues are obtained, so that cartilages are effectively and quickly repaired. The invention also discloses a preparation method of the coaxial electrostatic spinning fibrous scaffold. The preparation method comprises the steps of carrying out coaxial electrostatic spinning by using an aliphatic polyester shell solution and a polyvinylpyrrolidone core solution containing the recombinant human TGF-beta1 in the windless environment to prepare a fibrous scaffold; coupling the BMSC affinity peptides on the surface of the fibrous scaffold, thus obtaining a nanoscale coaxial electrostatic spinning fibrous scaffold. The method is simple, is easy to control and has relatively strong practicability.
Description
Technical field
The present invention relates to bioengineered tissue field, particularly a kind of coaxial electrostatic spinning fibrous framework and preparation method thereof.
Background technology
Fibrous framework is a kind of bioengineered tissue support, because it has the nanoscale structures close with n cell epimatrix, construction features that can bionic extracellular matrix, has the effects such as the Growth of Cells of support, guide tissue regeneration, control organizational structure and the delivery of biologically active factor.Because material and the structure of fibrous framework are the principal elements that affects its function, so very important to the preparation of fibrous framework.Utilize the prepared nano-scale fiber of coaxial electrostatic spinning technology not only to there is higher specific surface area and porosity, also there is the structure similar with extracellular matrix, can effectively be applied in organizational project method reparation cartilage injury process.Repair in cartilage injury's process in organizational project method, the fibrous framework that is placed in cartilage defect district will be raised mesenchymal stem cells MSCs (Bone Marrow Derived Mesenchymal Stem Cells, BMSC), BMSC grows in this fibrous framework, and to becoming the differentiation of cartilage direction, guiding cartilage tissue regeneration, promotes repair of cartilage.
Prior art is used the chloroformic solution of polycaprolactone as shell solution, the chloroformic solution of protein-Polyethylene Glycol (PEG) is as sandwich layer solution, by adopting coaxial electrostatic spinning technology to prepare the coaxial electrostatic spinning fibrous framework with the nanoscale structures close with n cell epimatrix, this coaxial electrostatic spinning fibrous framework comprises polycaprolactone shell and Polyethylene Glycol sandwich layer.
Realizing in process of the present invention, inventor finds that prior art at least exists following problem:
Polycaprolactone shell in the coaxial electrostatic spinning fibrous framework of preparing due to prior art is hydrophobicity, be difficult in conjunction with BMSC, the ability that the less and BMSC of BMSC that it is raised in cartilage defect district becomes the differentiation of cartilage direction a little less than, cause neocartilage to be organized less, be unfavorable for that cartilage repairs effectively rapidly.
Summary of the invention
The weak problem of ability that BMSC is less and BMSC becomes cartilage direction to break up of raising in cartilage defect district in order to solve prior art coaxial electrostatic spinning fibrous framework, the embodiment of the present invention provides a kind of coaxial electrostatic spinning fibrous framework.Described technical scheme is as follows:
On the one hand, the embodiment of the present invention provides a kind of coaxial electrostatic spinning fibrous framework, described coaxial electrostatic spinning fibrous framework comprises aliphatic polyester shell and polyvinylpyrrolidone sandwich layer, described aliphatic polyester shell surface coupling has mesenchymal stem cells MSCs affinity peptide, and in described polyvinylpyrrolidone sandwich layer, load has rhTGF-BETA-β 1.
Particularly, as preferably, described coaxial electrostatic spinning fibrous framework also comprises bovine serum albumin, and described bovine serum albumin loads in described polyvinylpyrrolidone sandwich layer.
Particularly, as preferably, in described aliphatic polyester shell, aliphatic polyester is selected from least one in polycaprolactone, polylactic acid, Poly(D,L-lactide-co-glycolide, poly-(lactic acid-hexanol) copolymer.
Particularly, as preferably, in aliphatic polyester shell in aliphatic polyester, polyvinylpyrrolidone sandwich layer polyvinylpyrrolidone and described bovine serum albumin quality than being 1-1.5:0.35-0.55:0.01-0.015.
Particularly, as preferably, the purity of described mesenchymal stem cells MSCs affinity peptide is more than or equal to 95%.
On the other hand, the embodiment of the present invention also provides a kind of preparation method of coaxial electrostatic spinning fibrous framework, and described method comprises:
Prepare aliphatic polyester shell solution: aliphatic polyester is dissolved in organic solvent, is stirred to completely and dissolves, obtain described aliphatic polyester shell solution;
The polyvinylpyrrolidone sandwich layer solution that preparation contains rhTGF-BETA-β 1: polyvinylpyrrolidone is dissolved in organic solvent, be stirred to completely and dissolve, obtain described polyvinylpyrrolidone sandwich layer solution, in described polyvinylpyrrolidone sandwich layer solution, add rhTGF-BETA-β 1, be stirred to mix homogeneously, described in obtaining, contain the polyvinylpyrrolidone sandwich layer solution of rhTGF-BETA-β 1;
Without under wind environment, respectively by described aliphatic polyester shell solution and described in contain rhTGF-BETA-β 1 polyvinylpyrrolidone sandwich layer solution inject shell solution injector and sandwich layer solution injector, carry out coaxial electrostatic spinning, prepare the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1;
The surface of the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1 described in mesenchymal stem cells MSCs affinity peptide is coupled to, obtains described coaxial electrostatic spinning fibrous framework;
Preparing aliphatic polyester shell solution organic solvent used is same with the polyvinylpyrrolidone sandwich layer solution organic solvent used that preparation contains rhTGF-BETA-β 1.
Particularly, as preferably, described method also comprises: in the process of the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 in preparation, in the described polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1, add bovine serum albumin.
Particularly, as preferably, the concentration of described aliphatic polyester shell solution is 0.14g/ml-0.16g/ml, and the concentration of described polyvinylpyrrolidone sandwich layer solution is 0.065g/ml-0.075g/ml.
Particularly, as preferably, described organic solvent is selected from least one in trifluoroethanol, formic acid, hexafluoroisopropanol, chloroform, ethanol.
Particularly, as preferably, the operating parameter of described coaxial electrostatic spinning is: spinning voltage is 10.5-10.8Kv; Spinning head is 18-20cm to the distance of collecting board; The fltting speed of aliphatic polyester shell solution is 0.20-0.22 ml/hour; The fltting speed of the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 is 0.13-0.15 ml/hour; The syringe needle internal diameter of shell solution injector is 0.89-0.91 millimeter; The syringe needle internal diameter of sandwich layer solution injector is 0.40-0.42 millimeter.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is:
The embodiment of the present invention provides a kind of coaxial electrostatic spinning fibrous framework, and it comprises that coupling has the aliphatic polyester shell of mesenchymal stem cells MSCs affinity peptide and load to have the polyvinylpyrrolidone sandwich layer of rhTGF-BETA-β 1.By the combination of aliphatic polyester shell and polyvinylpyrrolidone sandwich layer, not only make this fibrous framework there is the feature of safety non-toxic and biocompatibility excellence, also make this fibrous framework provide good biological platform for load slow releasing bioactivity factor.On the basis of this biology platform, by mesenchymal stem cells MSCs affinity peptide being coupled to aliphatic polyester shell surface, can strengthen the combination of BMSC and fibrous framework, impel this fibrous framework to raise the more BMSC of volume in cartilage defect district; By rhTGF-BETA-β 1 is loaded in polyvinylpyrrolidone sandwich layer, can promote this more BMSC one-tenth cartilage direction differentiation of volume, and then obtain more neocartilage tissue, cartilage is repaired effectively rapidly.
The embodiment of the present invention also provides a kind of preparation method of coaxial electrostatic spinning fibrous framework, by without under wind environment, the polyvinylpyrrolidone sandwich layer solution that uses aliphatic polyester shell solution and contain rhTGF-BETA-β 1 carries out coaxial electrostatic spinning, prepares the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1; And mesenchymal stem cells MSCs affinity peptide is coupled to the surface of aliphatic polyester shell in above-mentioned fibrous framework, obtain the coaxial electrostatic spinning fibrous framework with the nanoscale structures close with n cell epimatrix that the present invention expects.This support safety non-toxic, specific surface area and porosity are higher, and have the ability of raising BMSC and promoting the differentiation of BMSC one-tenth cartilage direction.The inventive method is simple, easy to control, and practicality is stronger.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the preparation flow figure of the coaxial electrostatic spinning fibrous framework that provides of the embodiment of the present invention;
Fig. 2 is the preparation flow figure of the coaxial electrostatic spinning fibrous framework that provides of further embodiment of this invention;
Fig. 3 is the preparation process schematic diagram of the coaxial electrostatic spinning fibrous framework that provides of further embodiment of this invention;
Fig. 4 is the transmission electron microscope picture of the coaxial electrostatic spinning fibrous framework that provides of further embodiment of this invention;
Fig. 5 is the scanning electron microscope (SEM) photograph of the coaxial electrostatic spinning fibrous framework that provides of further embodiment of this invention;
Fig. 6 is the diameter Distribution figure of the coaxial electrostatic spinning fibrous framework that provides of further embodiment of this invention;
Fig. 7 a, Fig. 7 b, Fig. 7 c and Fig. 7 d are the ability schematic diagrams that coaxial electrostatic spinning fibrous framework that further embodiment of this invention provides is raised BMSC;
Fig. 8 a, Fig. 8 b and Fig. 8 c are the slow-release capability schematic diagram of the coaxial electrostatic spinning fibrous framework that provides of further embodiment of this invention to rhTGF-β 1;
Fig. 9 a, Fig. 9 b, Fig. 9 c and Fig. 9 d are that the coaxial electrostatic spinning fibrous framework that further embodiment of this invention provides is urged the ability schematic diagram that BMSC becomes cartilage direction to break up.
Wherein, 1 shell solution injector,
2 sandwich layer solution injectors,
3 spinning heads,
4 collecting boaries,
5 coaxial electrostatic spinning fibrous frameworks,
51 aliphatic polyester shells,
52 polyvinylpyrrolidone sandwich layers,
53 rhTGF-β 1 factors,
6 hollow form coaxial electrically spun silk fiber supports,
7 do not contain the coaxial electrostatic spinning fibrous framework of E7 polypeptide and rhTGF-β 1,
The 8 coaxial electrostatic spinning fibrous frameworks containing E7 polypeptide,
The 9 coaxial electrostatic spinning fibrous frameworks containing E7 polypeptide and rhTGF-β 1.
The specific embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.
The embodiment of the present invention provides a kind of coaxial electrostatic spinning fibrous framework 5, this fibrous framework comprises aliphatic polyester shell 51 and polyvinylpyrrolidone sandwich layer 52, the surperficial coupling of this aliphatic polyester shell 51 has mesenchymal stem cells MSCs affinity peptide, and in this polyvinylpyrrolidone sandwich layer 52, load has rhTGF-BETA-β 1.
The embodiment of the present invention provides a kind of coaxial electrostatic spinning fibrous framework 5, and it comprises that coupling has the aliphatic polyester shell 51 of mesenchymal stem cells MSCs affinity peptide and load to have the polyvinylpyrrolidone sandwich layer 52 of rhTGF-BETA-β 1.Combination by aliphatic polyester shell 51 with polyvinylpyrrolidone sandwich layer 52, not only make this fibrous framework there is the feature of safety non-toxic and biocompatibility excellence, also make this fibrous framework provide good biological platform for load slow releasing bioactivity factor.On the basis of this biology platform, by mesenchymal stem cells MSCs affinity peptide being coupled to aliphatic polyester shell 51 surfaces, can strengthen the combination of BMSC and fibrous framework, impel this fibrous framework to raise the more BMSC of volume in cartilage defect district; By rhTGF-BETA-β 1 is loaded in polyvinylpyrrolidone sandwich layer 52, can promote this more BMSC one-tenth cartilage direction differentiation of volume, and then obtain more neocartilage tissue, cartilage is repaired effectively rapidly.
The embodiment of the present invention provides a kind of coaxial electrostatic spinning fibrous framework 5, this fibrous framework comprises aliphatic polyester shell 51 and polyvinylpyrrolidone sandwich layer 52, the surperficial coupling of this aliphatic polyester shell 51 has mesenchymal stem cells MSCs affinity peptide, and in this polyvinylpyrrolidone sandwich layer 52, load has rhTGF-BETA-β 1 and bovine serum albumin.
Because polyvinylpyrrolidone is a kind of water-soluble pharmaceutical intermediate and pharmaceutical adjuvant of safety non-toxic, can dissolve each other with many kinds of substance or compound, make coaxial electrostatic spinning fibrous framework 5 safety non-toxics and there is excellent biocompatibility; Because the N-H of polyvinylpyrrolidone or O-H bond energy and multi-medicament and/or bioactie agent form intermolecular association, and by release time and the action intensity of this association control medicine and/or bioactie agent, extend this medicine and/or the bioactie agent emission and absorption time in vivo, the function that can give 5 loads of coaxial electrostatic spinning fibrous framework slow releasing pharmaceutical and/or bioactie agent.Based on the above, the embodiment of the present invention is used the sandwich layer of polyvinylpyrrolidone material as coaxial electrostatic spinning fibrous framework 5.
For bioactie agent, rhTGF-BETA-β 1(hereinafter to be referred as: be rhTGF-β 1) to promote BMSC to become the standard biological factor of cartilage direction differentiation, so the preferred rhTGF-β 1 of the embodiment of the present invention.Be understandable that also other can be had and promote BMSC to become the bioactie agent of cartilage direction differentiation capability to load in polyvinylpyrrolidone sandwich layer 52.Because the biological half-life of rhTGF-β 1 is shorter, easily run off, the embodiment of the present invention by rhTGF-β 1 load (i.e. parcel) in polyvinylpyrrolidone sandwich layer 52 structures of coaxial electrically spun silk fiber support, to prevent that it runs off, guarantee that it can promote this more BMSC one-tenth cartilage direction differentiation of volume.Further; in order to guarantee that rhTGF-β 1 has good biological activity within the longer time; the embodiment of the present invention also will add polyvinylpyrrolidone sandwich layer 52 as the bovine serum albumin of protein protective agent and stabilizing agent, to help the biological activity that keeps rhTGF-β 1.
Particularly, as preferably, in aliphatic polyester shell 51, aliphatic polyester is selected from least one in polycaprolactone, polylactic acid, Poly(D,L-lactide-co-glycolide, poly-(lactic acid-hexanol) copolymer.
The requirement of Biodegradable, mechanical performance and processing characteristics based on to shell, the Shell Materials of embodiment of the present invention fibrous framework is selected at least one in polycaprolactone, polylactic acid, Poly(D,L-lactide-co-glycolide, poly-(lactic acid-hexanol) copolymer.And polycaprolactone due to have good biocompatibility and biological degradability, with the good advantage such as the compatibility, good solvent solubility of other high molecular polymer, the preferred polycaprolactone of the embodiment of the present invention is as Shell Materials, thereby gives the support performance of prepared coaxial electrostatic spinning fibrous framework 5 excellences.
Particularly, in aliphatic polyester shell in aliphatic polyester, polyvinylpyrrolidone sandwich layer polyvinylpyrrolidone and bovine serum albumin quality than being 1-1.5:0.35-0.55:0.01-0.015.
In order to control preferably the structure of prepared coaxial electrostatic spinning fibrous framework 5, make it have higher specific surface area and porosity, make the load capacity of rhTGF-BETA-β 1 and bovine serum albumin and slow-release capability in equilibrium point preferably, the mass ratio of aliphatic polyester, polyvinylpyrrolidone and bovine serum albumin is controlled at 1-1.5:0.35-0.55:0.01-0.015 by the embodiment of the present invention simultaneously.As preferably, the mass ratio of aliphatic polyester, polyvinylpyrrolidone and bovine serum albumin is 1:0.35:0.015.Be understandable that, in coaxial electrostatic spinning fibrous framework 5 provided by the invention, the amount of mesenchymal stem cells MSCs affinity peptide is controlled at it and is just evenly distributed on fully aliphatic polyester shell 51 and is advisable; The amount of rhTGF-BETA-β 1 is controlled at it and is just evenly distributed in bovine serum albumin and is advisable fully.
Particularly, as preferably, the purity of mesenchymal stem cells MSCs affinity peptide is more than or equal to 95%.
For the ability that makes the fibrous framework that the embodiment of the present invention provides raise more BMSC in cartilage defect district, improve this fibrous framework to raise BMSC, the purity of the preferred mesenchymal stem cells MSCs affinity peptide of the embodiment of the present invention is more than or equal to 95%.More specifically, this mesenchymal stem cells MSCs affinity peptide is a kind of functional BMSC specificity affinity peptide being made up of 7 aminoacid: E7 polypeptide.
Be understandable that, in order to guarantee that BMSC becomes the ability of cartilage direction differentiation, the purity of rhTGF-BETA-β 1 also should be controlled in the scope that is more than or equal to 95%.
As shown in Figure 1, the embodiment of the present invention provides a kind of preparation method of coaxial electrostatic spinning fibrous framework 5, comprising:
Step 101: prepare aliphatic polyester shell solution: aliphatic polyester is dissolved in organic solvent, is stirred to completely and dissolves, obtain aliphatic polyester shell solution.
Step 102: the polyvinylpyrrolidone sandwich layer solution that preparation contains rhTGF-BETA-β 1: polyvinylpyrrolidone is dissolved in same organic solvent, be stirred to completely and dissolve, obtain polyvinylpyrrolidone sandwich layer solution, in this polyvinylpyrrolidone sandwich layer solution, add rhTGF-BETA-β 1, be stirred to mix homogeneously, obtain the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1.
Step 103: without under wind environment, respectively aliphatic polyester shell solution and the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 are injected to shell solution injector 1 and sandwich layer solution injector 2, carry out coaxial electrostatic spinning, prepare the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1.
Step 104: mesenchymal stem cells MSCs affinity peptide is coupled to the surface of the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1, obtains coaxial electrostatic spinning fibrous framework 5.
Wherein, the solution organic solvent used of preparing the polyvinylpyrrolidone sandwich layer 52 that the solution of aliphatic polyester shell 51 organic solvent used and preparation contain rhTGF-BETA-β 1 is same.
The embodiment of the present invention also provides a kind of preparation method of coaxial electrostatic spinning fibrous framework 5, by without under wind environment, the solution of the polyvinylpyrrolidone sandwich layer 52 that uses the solution of aliphatic polyester shell 51 and contain rhTGF-BETA-β 1 carries out coaxial electrostatic spinning, prepares the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1; And mesenchymal stem cells MSCs affinity peptide is coupled to the surface of aliphatic polyester shell 51 in above-mentioned fibrous framework, obtain the nanoscale coaxial electrostatic spinning fibrous framework 5 that the present invention expects.This support safety non-toxic, specific surface area and porosity are higher, and have the ability of raising more BMSC and promoting the differentiation of BMSC one-tenth cartilage direction.The inventive method is simple, easy to control, and practicality is stronger.
Embodiment 4
As shown in Figure 2, the embodiment of the present invention provides a kind of preparation method of coaxial electrostatic spinning fibrous framework 5, comprising:
Step 201: prepare aliphatic polyester shell solution: aliphatic polyester is dissolved in organic solvent, is stirred to completely and dissolves, obtain the solution of aliphatic polyester shell 51, the concentration of controlling this aliphatic polyester shell solution is 0.14g/ml-0.16g/ml.
Coaxial electrostatic spinning crosses range request shell and sandwich layer solution has suitable concentration, is understandable that, concentration and viscosity are directly proportional.In order to guarantee that aliphatic polyester shell solution has enough large surface tension and electric field force to balance each other, and form stable taylor cone by the spinning nozzle place that acts on of this equilibrant, and then guarantee that prepared fibrous framework has the structure of stable uniform, the embodiment of the present invention is can spinning in the situation that, the concentration of controlling this aliphatic polyester shell solution is 0.14g/ml-0.16g/ml, preferably 0.15g/ml.
Step 202: the polyvinylpyrrolidone sandwich layer solution that preparation contains rhTGF-BETA-β 1: polyvinylpyrrolidone is dissolved in organic solvent, be stirred to completely and dissolve, obtain polyvinylpyrrolidone sandwich layer solution, the concentration of controlling this polyvinylpyrrolidone sandwich layer solution is 0.065g/ml-0.075g/ml, in this polyvinylpyrrolidone sandwich layer solution, add rhTGF-BETA-β 1 and bovine serum albumin, be stirred to mix homogeneously, obtain the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1.
Wherein, in order to improve the compatibility of shell and sandwich layer in fibrous framework, in step 201, preparing the polyvinylpyrrolidone sandwich layer solution organic solvent used that in aliphatic polyester shell solution organic solvent used and step 202, preparation contains rhTGF-BETA-β 1 is same.
Because the concentration of sandwich layer solution can not too greatly again can not be too little, because in the situation that other conditions are constant, the concentration of sandwich layer solution is excessive, what shell solution produced at interface place can not overcome self viscoelastic power to the viscous friction of sandwich layer solution, and then good drawing-off formation composite injection thread; The viscosity of sandwich layer solution is too small, can not form continuous stable injection thread, causes the unstability of this coaxial electrostatic spinning process to increase.So the concentration of this polyvinylpyrrolidone sandwich layer solution of embodiment of the present invention control is 0.065g/ml-0.075g/ml, preferably 0.07g/ml.
Step 203: without under wind environment, respectively institute's aliphatic polyester shell solution and the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 are injected to shell solution injector 1 and sandwich layer solution injector 2, carry out coaxial electrostatic spinning, prepare the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1.
As shown in Figure 3, in coaxial electrostatic spinning process, shell solution and sandwich layer solution are injected through shell solution injector 1 and sandwich layer solution injector 2 respectively, at same spinning head, 3 places form fiber jet, and by the preferred aluminium foil of collecting board 4() collect, obtain coaxial electrostatic spinning fibrous framework.This fiber jet is before uncured, and its structure is easy to be subject to the impact of extraneous factor, and in order to guarantee structure and the length stable homogeneous of obtained fibrous framework, the embodiment of the present invention is without carrying out coaxial electrostatic spinning operation under wind environment.
Step 204: mesenchymal stem cells MSCs affinity peptide is coupled to the surface of the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1, obtains coaxial electrostatic spinning fibrous framework 5.
Wherein, the above-mentioned surface that mesenchymal stem cells MSCs affinity peptide is coupled to the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1 refers to the surface that mesenchymal stem cells MSCs affinity peptide is coupled to aliphatic polyester shell 51 in this fibrous framework.
Particularly, above-mentioned organic solvent is selected from least one in trifluoroethanol, formic acid, hexafluoroisopropanol, chloroform, ethanol.
In order to effectively reduce the interfacial tension between shell and sandwich layer solution, make shell solution drive sandwich layer solution to carry out better drawing-off, form good composite injection thread; And guarantee that fibrous framework center core layer-shell border of obtaining is clearly demarcated, structure is more perfect, the embodiment of the present invention selects at least one in trifluoroethanol, formic acid, hexafluoroisopropanol, chloroform, ethanol as electrostatic spinning organic solvent, preferably trifluoroethanol.
Particularly, as preferably, the operating parameter of above-mentioned coaxial electrostatic spinning is: spinning voltage is 10.5-10.8Kv; Spinning head 3 is 18-20cm to the distance of collecting board 4; The fltting speed of aliphatic polyester shell solution is 0.20-0.22 ml/hour; The fltting speed of the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 is 0.13-0.15 ml/hour; The syringe needle internal diameter of shell solution injector 1 is 0.89-0.91 millimeter; The syringe needle internal diameter of sandwich layer solution injector 2 is 0.40-0.42 millimeter.
In coaxial electrostatic spinning process, very crucial to the control of its operating parameter.Particularly, the flow velocity of controlling sandwich layer and shell solution is very crucial for obtaining the fibrous framework of the good core-shell structure of form.In order to prevent that sandwich layer solution flow rate is too fast to such an extent as to break through the parcel of shell solution, to form stable composite injection thread; Carry out independent electrostatic spinning for fear of shell solution, obtain the fibrous framework of the continuous core-shell structure of the good even thickness of form, the fltting speed of polycaprolactone shell solution is controlled at 0.20-0.22 ml/hour by the embodiment of the present invention, preferably 0.21 ml/hour; The fltting speed of the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 is controlled to 0.13-0.15 ml/hour, preferably 0.14 ml/hour.
For spinning voltage, in the time that added voltage is different, for breaking surface tension and electric field equilibrium of forces, the drop on capillary tube top will produce different surface configurations, the liquid droplets that impact produces subsequently and distribution situation, fibre morphology and its size of current of conducting of thread size.In the time that applied voltage is lower, initial injection point is dashed forward in the outside of shower nozzle, sprays thread and also will produce in drop is most advanced and sophisticated, and now the diameter of drop is greater than the aperture of spraying syringe needle, and the nanofiber of gained is thinner, and knot pearl is less; After voltage increases, liquid droplets sprays retraction in syringe needle, makes to spray thread and is produced by needle tip thereupon, and the line density of gained nanofiber and knot pearl density also have corresponding increase; Voltage continues to be increased to critical, and thread will be gone out by the direct sharp spray of syringe needle inwall, no longer forms and sprays thread, and only form liquid droplets, makes to connect pearl density and sharply rises.Based on more than, spinning voltage is controlled at 10.5-10.8Kv by the embodiment of the present invention, preferably 10.6Kv, thereby guarantee that the fibrous framework obtaining is nanoscale structures, and smooth without knot pearl.
Distance for spinning head 3 to collecting board 4, on the one hand, in order to prevent that prepared fibrous framework from too disperseing, it is long that collecting board 4 is collected certain thickness fibrous framework required time; On the other hand, in order to prevent that obtained fibrous framework from forming band shape or beading structure, embodiment of the present invention control spinning head 3 is 18-20cm to the distance of collecting board 4, preferably 20cm.
Further, in order to make the core-shell structure of prepared fibrous framework clear and even, the syringe needle internal diameter of shell solution injector 1 is controlled at 0.89-0.91 millimeter by the embodiment of the present invention, preferably 0.90 millimeter; The syringe needle internal diameter of sandwich layer solution injector 2 is controlled to 0.40-0.42 millimeter, preferably 0.41 millimeter.
1) the coaxial electrostatic spinning fibrous framework that preparation contains rhTGF-β 1:
Compound concentration is that the trifluoroethanol solution of polycaprolactone of 0.15g/ml is as shell solution, compound concentration is that the trifluoroethanol solution of polyvinylpyrrolidone of 0.07g/ml is as sandwich layer solution, and add rhTGF-β 1 and bovine serum albumin in this sandwich layer solution, stir, prepare the polyvinylpyrrolidone sandwich layer solution containing rhTGF-β 1, the concentration of controlling rhTGF-β 1 in this solution is 5 μ g/ml; The concentration of bovine serum albumin is 0.002g/ml.
As shown in Figure 3, without under wind environment, add in shell solution injector 1 and sandwich layer solution injector 2 by the polycaprolactone shell solution of above-mentioned preparation with containing the polyvinylpyrrolidone sandwich layer solution of rhTGF-β 1 respectively, carry out coaxial electrostatic spinning, prepare the coaxial electrostatic spinning fibrous framework containing rhTGF-β 1.Particularly, this support is uniform cylindrical fiber roughly, and the shell of this support is polycaprolactone; The sandwich layer of this support is the polyvinylpyrrolidone containing rhTGF-β 1 and bovine serum albumin.
In above-mentioned coaxial electrostatic spinning process, control spinning voltage is 10.6Kv; Spinning head 3 is 20cm to the distance of collecting board 4; The fltting speed of polycaprolactone shell solution is 0.21 ml/hour; The fltting speed that contains the polyvinylpyrrolidone sandwich layer solution of rhTGF-β 1 is 0.14 ml/hour; The syringe needle internal diameter of shell solution injector 1 is 0.90 millimeter; The syringe needle internal diameter of sandwich layer solution injector 2 is 0.41 millimeter.
2) E7 polypeptide is coupled on the coaxial electrostatic spinning fibrous framework that contains rhTGF-β 1
A) the coaxial electrostatic spinning fibrous framework that contains rhTGF-β 1 is put into the hole of 12 orifice plates, and to add 1 ml concn be 1 of 0.1g/ml, 6-hexamethylene diamine solution, 37 ℃ of reaction 1h.After completion of the reaction, deionized water carefully washs this support 2 times.
B) in each hole of 12 orifice plates, add the 4-that 400 μ l concentration are 2mg/mL (N-maleimide methyl) cyclohexane extraction-1-carboxylic acid sulfonic group succinimide ester sodium, room temperature reaction 1h.
C) by Fluorescein isothiocyanate (fluorescein isothiocyanate, FITC) the E7 polypeptide of labelling is dissolved in phosphate buffered solution, being mixed with concentration is the phosphate buffered solution of the E7 polypeptide of 0.1mg/ml, the phosphate buffered solution of injecting 400 these E7 polypeptide of μ l at each hole of above-mentioned 12 orifice plates infiltrates above-mentioned fibrous framework, 37 ℃ of reaction 2h.React complete, the above-mentioned each support of deionized water rinsing 2 times, obtains the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1.By this fibrous framework vacuum lyophilization ,-20 ℃ of preservations.
Utilize the fibrous framework of the above-mentioned preparation of transmission electron microscope observing, as shown in Figure 4, coaxial electrostatic spinning fibrous framework 5 prepared by the embodiment of the present invention " shell-core " double-decker significantly clearly as seen, and the structural integrity homogeneous of aliphatic polyester shell 51 and polyvinylpyrrolidone sandwich layer 52, illustrates that load has the polyvinylpyrrolidone sandwich layer 52 of bovine serum albumin and rhTGF-β 1 successfully to be wrapped up by aliphatic polyester shell 51.Be understandable that, in the embodiment of the present invention, coaxial electrostatic spinning fibrous framework 5 is same with the coaxial electrostatic spinning fibrous framework 9 that contains E7 polypeptide and rhTGF-β 1.
Utilize the prepared fibrous framework of scanning electron microscopic observation, as shown in Figure 5, the fibre morphology homogeneous of coaxial electrostatic spinning fibrous framework 5 prepared by the embodiment of the present invention, smooth, form without knot pearl.And the diameter of fibrous framework prepared by the embodiment of the present invention is mainly distributed in 600-700 nanometer left and right, belongs to nanoscale structures, with reference to Fig. 6.Visible, coaxial electrostatic spinning fibrous framework 5 structural parameters that the embodiment of the present invention provides are all better, can imitate natural extracellular matrix structure, are applicable to the growth of cell in organizational project, reached the application requirements of bioengineered tissue.
Respectively the E7 polypeptide in this fibrous framework and bovine serum albumin are carried out to fluorescent labeling, in the fibrous framework of preparing by the confocal microscopy embodiment of the present invention, E7 polypeptide and bovine serum albumin are evenly and continuous distribution.Visible, fibrous framework prepared by the embodiment of the present invention success coupling E7 polypeptide, possessed the ability that specificity is raised BMSC.
The ability that the embodiment of the present invention is raised BMSC to coaxial electrostatic spinning fibrous framework 5 detects, and detailed process is as follows:
1) according to the operating condition in above-described embodiment 5, under same condition, prepare respectively containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide, not containing the coaxial electrostatic spinning fibrous framework 7 of E7 polypeptide and rhTGF-β 1.Be understandable that, also comprise bovine serum albumin containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, below repeat no longer one by one.
2) respectively the BMSC kind of rat is planted to the above-mentioned coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide, not on the coaxial electrostatic spinning fibrous framework 7 containing E7 polypeptide and rhTGF-β 1, all with analyzing culture medium culturing 1, 3, 5, 7 hours, detect the adhesion number of BMSC on above-mentioned each fibrous framework, as shown in accompanying drawing 7a, compare the not coaxial electrostatic spinning fibrous framework 7 containing E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, containing the BMSC that sticks greater number on the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide, wherein, the BMSC sticking on coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 is more.Visible, the embodiment of the present invention, by E7 polypeptide and rhTGF-β 1 are loaded on coaxial electrostatic spinning fibrous framework 5, has obviously improved the ability that fibrous framework is raised BMSC.
Above-mentioned each support was cultivated respectively after 24 hours, the cytoskeleton of BMSC is dyeed with the phalloidin of rhodamine labelling, utilize the extended configuration of microscopic examination BMSC on above-mentioned each support, as shown in accompanying drawing 7b, compare the not coaxial electrostatic spinning fibrous framework 7 containing E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, obviously better (wherein, scheme mellow lime white portion and represent the BMSC of distribution) containing the extended configuration of the BMSC raising on the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide.Wherein, the extended configuration optimum of the BMSC raising on the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1.Visible, the embodiment of the present invention, by E7 polypeptide and rhTGF-β 1 are loaded on coaxial electrostatic spinning fibrous framework 5, has not only obviously improved the ability that fibrous framework is raised BMSC, and provides platform preferably for the growth of BMSC.
Above-mentioned each support was cultivated respectively after 7 and 14 days, is detected the DNA content of BMSC in above-mentioned each support, and at electric Microscopic observation BMSC the growth conditions on above-mentioned each support.As accompanying drawing 7c and accompanying drawing 7d(wherein in Electronic Speculum figure finer and close region be the BMSC of distribution) as shown in, compare the not coaxial electrostatic spinning fibrous framework 7 containing E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, more excellent containing the growth conditions of the BMSC raising on the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide, wherein, the growth conditions optimum of the BMSC raising on the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1.Visible, the embodiment of the present invention, by E7 polypeptide and rhTGF-β 1 are loaded on coaxial electrostatic spinning fibrous framework 5, can not only be raised more BMSC, and has obviously improved the growth conditions of the BMSC being raised.
Wherein, in the embodiment of the present invention, the constituent of analysis culture medium used is: DMEM(dulbecco's modified eagle medium) culture fluid, 100nm dexamethasone, the ascorbic acid usp/bp of 50 μ g/mL, the Sodium Pyruvate of 100 μ g/mL, the proline of 40 μ g/mL, the penicillin of 100U/mL and streptomycin, ITS+Premix somatomedin (it comprises the insulin of 6.25 μ g/mL, the transferrin of 6.25 μ g/mL, Monohydrated selenium dioxide, the linoleic acid of 5.35 μ g/mL and the bovine serum albumin of 1.25 μ g/mL of 6.25 μ g/mL).
The embodiment of the present invention is tested the slow-release capability of rhTGF-β 1 factor 53 the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 of above-mentioned preparation, and detailed process is as follows:
Get the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 of 30 milligrams of above-mentioned preparations, and with the phosphate buffer immersion of 1.5 milliliters, obtain soak, be placed in 37 ℃.Every day, soak 500 microlitres were got in timing, as sample, then with fresh phosphate buffer, soak were added to 1.5 milliliters.Repeat aforesaid operations to the 21 days, the ELISA test kit of use rhTGF-β 1 detects the content of rhTGF-β 1 in institute's label taking basis.Calculate the cumulative release amount of rhTGF-β 1 according to cumulative release amount computing formula, to reflect the above-mentioned slow-release capability containing the rhTGF-β 1 in the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1.Wherein cumulative release amount computing formula is:
Cumulative release amount (%)=100 × Mt/Mn
Mt is illustrated in time point t support and discharges the quality of rhTGF-β 1, and Mn represents the quality (in the embodiment of the present invention, Mn gets 6) of the rhTGF-β 1 carrying in support.
As shown in accompanying drawing 8a and accompanying drawing 8b, the release of succeeding in the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 in the present invention of rhTGF-β 1 factor 53, after rhTGF-β 1 factor 53 discharges, should become hollow form coaxial electrically spun silk fiber support 6 containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1.Visible, the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 that the embodiment of the present invention provides can effectively discharge it as the carrier of rhTGF-β 1 factor 53.As shown in accompanying drawing 8c, the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 that the embodiment of the present invention provides can be divided into two stages to the dispose procedure of rhTGF-β 1 factor 53, within 1-7 days, are release periods gradually of rhTGF-β 1 factor 53, within 8-21 days, are slow release periods of rhTGF-β 1 factor 53, slow release process stabilization.Visible, the coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 that the embodiment of the present invention provides has successfully carried out slow release to rhTGF-β 1 factor 53, and slow release effect is stable, and slow-release time is long.
The embodiment of the present invention respectively under above-mentioned same operation conditions, prepare containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, containing coaxial electrostatic spinning fibrous framework 8(first matched group of E7 polypeptide), not containing coaxial electrostatic spinning fibrous framework 7(the second matched group of E7 polypeptide and rhTGF-β 1) short BMSC becomes the ability of cartilage direction differentiation to test, specifically test process is as follows:
Respectively the BMSC kind of rat is planted containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide with not on the coaxial electrostatic spinning fibrous framework 7 containing E7 polypeptide and rhTGF-β 1, cultivate after 14 days with analyzing culture medium (analysis culture medium used is of the same race with embodiment 6), draw materials, detect as follows:
1) cartilage specific gene: the real-time polymerase chain reaction (Polymerase Chain Reaction, PCR) of Type Ⅱ collagen (collagen2, COL2), proteoglycan (Proteoglycan) gene detects:
Primer sequence used is as follows:
The forward primer of Type Ⅱ collagen: 5 '-ACGGCGGCTTCCACTTCAGC-3 ';
The reverse primer of Type Ⅱ collagen: 5 '-TTGCCGGCTGCTTCGTCCAG-3 ';
The forward primer of proteoglycan: 5 '-CATTCGCACGGGAGCAGCCA-3 ';
The reverse primer of proteoglycan: 5 '-TGGGGTCCGTGGGCTCACAA-3 ';
The forward primer of beta-actin: 5 '-CTAAGGCCAACCGTGAAAAG-3 ';
The reverse primer of beta-actin: 5 '-AACACAGCCTGGATGGCTAC-3 '.
Real-time PCR reactions operating condition is as follows:
7300 real-time PCR systems: 95 ℃ of degeneration 10 minutes;
40 circulations: at 95 ℃ 15 seconds, 60 ℃ of downward-extensions 1 minute
Solubility curve: at 95 ℃ 15 seconds, be progressively warming up to 95 ℃ after 30 seconds at 60 ℃ and unwind.
Δ CT experimental group=CT experimental group gene-CT experimental group internal reference;
Δ CT matched group=CT matched group gene-CT matched group internal reference;
Δ Δ CT=Δ CT experimental group-Δ CT matched group;
2
-Δ Δ CT=2
-(Δ CT experimental group-Δ CT matched group).
As shown in accompanying drawing 9a and 9b (wherein, expression in figure all represents to express multiple), compare the coaxial electrostatic spinning fibrous framework 7 that contains the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide and do not contain E7 polypeptide and rhTGF-β 1, the Type Ⅱ collagen of coaxial electrostatic spinning fibrous framework 9 and the gene expression amount of proteoglycan containing E7 polypeptide and rhTGF-β 1 that the embodiment of the present invention provides are higher, visible, the present invention can significantly promote BMSC to become the differentiation of cartilage direction by rhTGF-β 1 being loaded in fibrous framework, Regeneration and Repair for damaged cartilage has great importance.
2) measure proteoglycan is carried out to detection by quantitative by Dimethylmethylene blue (1,9-dimethylmethylene blue, DMMB) method, detailed process is as follows:
A) kind is implanted with to BMSCs containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide with do not cultivate after 14 days containing the coaxial electrostatic spinning fibrous framework 7 of E7 polypeptide and rhTGF-β 1, add respectively 1 milliliter of papain, at 60 ℃, hatch 24 hours.
B), respectively to the DMMB that adds 200 μ l in the every hole in 96 orifice plates, then get above-mentioned each support of 20ul and 18ul phosphate buffer and add in above-mentioned 96 orifice plates and mix with DMMB.
C) utilize microplate reader (525 nano wave length) to measure absorbance, calculate the content of proteoglycan in above-mentioned each support.
As shown in accompanying drawing 9c (wherein, the unit of proteoglycan content is microgram/support), compare the coaxial electrostatic spinning fibrous framework 7 that contains the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide and do not contain E7 polypeptide and rhTGF-β 1, the amount containing proteoglycan in the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1 that the embodiment of the present invention provides is higher, visible, the present invention can significantly promote BMSC to become the differentiation of cartilage direction by rhTGF-β 1 being loaded in fibrous framework, has great importance for the Regeneration and Repair of damaged cartilage.
3) immunofluorescence of Type Ⅱ collagen detects:
A) specimen is fixed: by plant be respectively implanted with BMSC containing the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1, containing the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide with do not cultivate after 14 days containing the coaxial electrostatic spinning fibrous framework 7 of E7 polypeptide and rhTGF-β 1, the paraformaldehyde room temperature that is 4% by concentration is fixed 10 minutes.
B) cell is permeabilized: cover above-mentioned each support, room temperature, rinsing 5 minutes and 3 times with penetrating liquid (phosphate tween buffer: containing the concentration TritonX-100 that is 0.3-0.5%, pH is 7.4).
C) closed supports: utilizing concentration is that 2% bovine serum albumin covers above-mentioned each support, wet box inner sealing 45 minutes at 37 ℃.
D) primary antibodie is hatched: add unlabelled anti-Type Ⅱ collagen antibody (1:200 dilution) to above-mentioned each support, 4 ℃ are spent the night.
E) rinsing: the above-mentioned each support of phosphate buffer rinsing 5 minutes and 3 times.
F) two anti-hatching: add two anti antibodys of rhodamine labelling to above-mentioned each support, 37 ℃ of wet box lucifuges 40 minutes.
G) utilize Hoechst33258 labeled cell core (Nuclei), rinsing, mounting, observes under Laser Scanning Confocal Microscope.
As shown in accompanying drawing 9d, after In vitro culture BMSC a period of time, compare the coaxial electrostatic spinning fibrous framework 7 that contains the coaxial electrostatic spinning fibrous framework 8 of E7 polypeptide and do not contain E7 polypeptide and rhTGF-β 1, it is more obvious containing BMSC propagation in the coaxial electrostatic spinning fibrous framework 9 of E7 polypeptide and rhTGF-β 1 that the embodiment of the present invention provides, and the synthetic quantity of Type Ⅱ collagen (COL2) is higher.Visible, the present invention can significantly promote BMSC to become the differentiation of cartilage direction by rhTGF-β 1 being loaded in fibrous framework, has great importance for the Regeneration and Repair of damaged cartilage.
In sum, coaxial electrostatic spinning fibrous framework 9 containing E7 polypeptide and rhTGF-β 1 prepared by the embodiment of the present invention is repaired the application advantage in field for as follows at cartilaginous tissue: 1) have nanoscale structures as support, can analog cell epimatrix structure, be beneficial to Growth of Cells; 2) seed cell: BMSC can be raised, and adhesion and the growth of BMSCs on support can be obviously improved and be beneficial to; 3) can secrete bioactie agent: rhTGF-β 1, has promoted BMSC to become the differentiation of cartilage direction significantly.Visible, the coaxial electrostatic spinning fibrous framework 5 that the embodiment of the present invention provides, by load E7 polypeptide and rhTGF-β 1, can be repaired damaged cartilage effectively, has stronger practicality and adaptability in repair of cartilage field.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. a coaxial electrostatic spinning fibrous framework, it is characterized in that, described coaxial electrostatic spinning fibrous framework comprises aliphatic polyester shell and polyvinylpyrrolidone sandwich layer, described aliphatic polyester shell surface coupling has mesenchymal stem cells MSCs affinity peptide, and in described polyvinylpyrrolidone sandwich layer, load has rhTGF-BETA-β 1.
2. coaxial electrostatic spinning fibrous framework according to claim 1, is characterized in that, described coaxial electrostatic spinning fibrous framework also comprises bovine serum albumin, and described bovine serum albumin loads in described polyvinylpyrrolidone sandwich layer.
3. coaxial electrostatic spinning fibrous framework according to claim 1, it is characterized in that, in described aliphatic polyester shell, aliphatic polyester is selected from least one in polycaprolactone, polylactic acid, Poly(D,L-lactide-co-glycolide, poly-(lactic acid-hexanol) copolymer.
4. coaxial electrostatic spinning fibrous framework according to claim 2, it is characterized in that, in aliphatic polyester shell in aliphatic polyester, polyvinylpyrrolidone sandwich layer polyvinylpyrrolidone and described bovine serum albumin quality than being 1-1.5:0.35-0.55:0.01-0.015.
5. coaxial electrostatic spinning fibrous framework according to claim 1, is characterized in that, the purity of described mesenchymal stem cells MSCs affinity peptide is more than or equal to 95%.
6. a preparation method for the coaxial electrostatic spinning fibrous framework described in claim 1-5 any one, is characterized in that, described method comprises:
Prepare aliphatic polyester shell solution: aliphatic polyester is dissolved in organic solvent, is stirred to completely and dissolves, obtain described aliphatic polyester shell solution;
The polyvinylpyrrolidone sandwich layer solution that preparation contains rhTGF-BETA-β 1: polyvinylpyrrolidone is dissolved in organic solvent, be stirred to completely and dissolve, obtain described polyvinylpyrrolidone sandwich layer solution, in described polyvinylpyrrolidone sandwich layer solution, add rhTGF-BETA-β 1, be stirred to mix homogeneously, described in obtaining, contain the polyvinylpyrrolidone sandwich layer solution of rhTGF-BETA-β 1;
Without under wind environment, respectively by described aliphatic polyester shell solution and described in contain rhTGF-BETA-β 1 polyvinylpyrrolidone sandwich layer solution inject shell solution injector and sandwich layer solution injector, carry out coaxial electrostatic spinning, prepare the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1;
The surface of the coaxial electrostatic spinning fibrous framework that contains rhTGF-BETA-β 1 described in mesenchymal stem cells MSCs affinity peptide is coupled to, obtains described coaxial electrostatic spinning fibrous framework;
Preparing aliphatic polyester shell solution organic solvent used is same with the polyvinylpyrrolidone sandwich layer solution organic solvent used that preparation contains rhTGF-BETA-β 1.
7. the preparation method of coaxial electrostatic spinning fibrous framework according to claim 6, it is characterized in that, described method also comprises: in the process of the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 in preparation, in the described polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1, add bovine serum albumin.
8. the preparation method of coaxial electrostatic spinning fibrous framework according to claim 6, it is characterized in that, the concentration of described aliphatic polyester shell solution is 0.14g/ml-0.16g/ml, and the concentration of described polyvinylpyrrolidone sandwich layer solution is 0.065g/ml-0.075g/ml.
9. the preparation method of coaxial electrostatic spinning fibrous framework according to claim 6, is characterized in that, described organic solvent is selected from least one in trifluoroethanol, formic acid, hexafluoroisopropanol, chloroform, ethanol.
10. according to the preparation method of the coaxial electrostatic spinning fibrous framework described in claim 6-9 any one, it is characterized in that, the operating parameter of described coaxial electrostatic spinning is: spinning voltage is 10.5-10.8Kv; Spinning head is 18-20cm to the distance of collecting board; The fltting speed of aliphatic polyester shell solution is 0.20-0.22 ml/hour; The fltting speed of the polyvinylpyrrolidone sandwich layer solution that contains rhTGF-BETA-β 1 is 0.13-0.15 ml/hour; The syringe needle internal diameter of shell solution injector is 0.89-0.91 millimeter; The syringe needle internal diameter of sandwich layer solution injector is 0.40-0.42 millimeter.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106730038A (en) * | 2017-01-12 | 2017-05-31 | 广东泰宝医疗器械技术研究院有限公司 | A kind of tunica fibrosa for tracheae soft tissue repair and preparation method thereof |
| CN109498837A (en) * | 2018-10-31 | 2019-03-22 | 武汉大学 | A method of combination coaxial electrostatic spinning and LBL self-assembly collaboration gradient discharge growth factor |
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| CN113584712A (en) * | 2021-09-01 | 2021-11-02 | 大连工业大学 | Method for preparing nano composite fiber membrane based on electrostatic spinning technology |
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| CN114525599A (en) * | 2022-03-17 | 2022-05-24 | 北京市创伤骨科研究所 | Bionic periosteum and preparation method and application thereof |
| CN114984295A (en) * | 2022-04-14 | 2022-09-02 | 广东云曌医疗科技有限公司 | Porous nano medical dressing and preparation method thereof |
| CN109172073B (en) * | 2018-09-03 | 2023-11-21 | 上海市第一人民医院 | Electrostatic spinning nanofiber membrane for controlling release of growth factors and esophageal tectorial membrane memory stent |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107625995B (en) * | 2017-08-18 | 2020-07-14 | 北京市创伤骨科研究所 | Multilayer coaxial fiber bone repair membrane material and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1727530A (en) * | 2005-07-26 | 2006-02-01 | 天津大学 | Superfine fiber membrane material in core/shell structure, and preparation method |
| CN103147165A (en) * | 2013-01-25 | 2013-06-12 | 四川大学 | Double-wall structured hollow ultrafine polymer fiber and preparation method thereof |
-
2014
- 2014-03-20 CN CN201410105964.2A patent/CN103893819B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1727530A (en) * | 2005-07-26 | 2006-02-01 | 天津大学 | Superfine fiber membrane material in core/shell structure, and preparation method |
| CN103147165A (en) * | 2013-01-25 | 2013-06-12 | 四川大学 | Double-wall structured hollow ultrafine polymer fiber and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 孙斌: "芯/壳结构超细纤维及其用作伤口敷料的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 袁晓燕: "生物医用电纺芯/壳结构超细纤维膜的研究", 《2010年全国高分子材料科学与工程研讨会》 * |
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