[go: up one dir, main page]

WO2013183976A1 - Patch destiné à la régénération tissulaire, comprenant un échafaudage tridimensionnel poreux fibreux - Google Patents

Patch destiné à la régénération tissulaire, comprenant un échafaudage tridimensionnel poreux fibreux Download PDF

Info

Publication number
WO2013183976A1
WO2013183976A1 PCT/KR2013/005094 KR2013005094W WO2013183976A1 WO 2013183976 A1 WO2013183976 A1 WO 2013183976A1 KR 2013005094 W KR2013005094 W KR 2013005094W WO 2013183976 A1 WO2013183976 A1 WO 2013183976A1
Authority
WO
WIPO (PCT)
Prior art keywords
patch
support
tissue
cells
stem cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2013/005094
Other languages
English (en)
Korean (ko)
Inventor
이승진
심인경
양영일
장양수
정미라
정혜진
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ewha Womans University
Original Assignee
Ewha Womans University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ewha Womans University filed Critical Ewha Womans University
Priority to US14/047,948 priority Critical patent/US20210308335A1/en
Publication of WO2013183976A1 publication Critical patent/WO2013183976A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F13/00—Bandages or dressings; Absorbent pads
    • A61F13/02—Adhesive bandages or dressings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/16—Compositions of unspecified macromolecular compounds the macromolecular compounds being biodegradable

Definitions

  • the present invention is a fibrous porous three-dimensional support; And a cell, a drug, a bioactive substance, or a combination thereof contained on or in the surface of the support, and a patch for tissue regeneration and a method of manufacturing the same.
  • the heart was known as a tissue that can not be recovered once damaged.
  • cardiomyocyte cell division and loss as a replacement of cardiomyocytes have been reported to occur slowly, but conventional medical treatments do not provide a way to increase the number of damaged cardiomyocytes.
  • patients with severe end-stage heart failure are unable to restore the function of the heart, so the only way is to use a heart transplant or ventricular assist device.
  • cell transplantation therapeutics using cells have emerged as an alternative for the recovery of damaged tissues.
  • Cells used for cell transplantation are myoblasts, cardiomyocytes, endothelial cells, fibroblasts, or stem cells that induce cardiovascular formation isolated from adult stem cells.
  • Such stem cell administration methods include intracardiac delivery of cardiac patients, direct injection into the myocardium surgically, injection into a coronary artery using a catheter, and systemic administration such as venous vascular injection. Method and the like.
  • bioretention of the injected cells is very low, and reports indicate that only about 10% of the injected cardiomyocytes affect the myocardial tissue regeneration due to the specificity of the heart. Therefore, in order to continuously regenerate myocardial tissue, the infusion rate of cardiomyocytes must be increased to the patient, and the patient's pain is also increased.
  • the actual number of cells to be transplanted is very small, so there is a problem that a high concentration of cells should be used for transplantation, and even though a high concentration of cells is transplanted, the cells remain in a poor environmental condition until they differentiate at the transplanted site, so the therapeutic effect is exceeded. There is a limit to the look.
  • the cell sheet refers to a single cell layer, and it is known that neonatal cardiomyocytes can be made into a single layered sheet and the sheets can be superimposed on up to three layers in vitro (Non-patent Document 11: FASEB J). 2006 Apr; 20 (6): 708-10). Recently, a variety of cell sheets have been produced by using a temperature sensitive culture plate coated with an electron beam onto a commercially available polystyrene culture plate surface of poly (N-isopropylacrylamide) (PIAAm).
  • PIAAm poly (N-isopropylacrylamide)
  • the production method of the cell sheet using the temperature sensitive culture dish is relatively stable when the initial culture is performed in a strict manner so as to be optimal for cultivation in the special culture dish, but it is customary in each facility.
  • the cell sheeting was difficult when the method which has been carried out by the above method was applied as it is.
  • the cell sheet is pressed by using a method of laminating cell sheets, and the cell survival rate is low, and due to the limitation of oxygen permeability, there is a problem in that the cell sheet can not be made thicker without angiogenesis.
  • a method of transplanting cells using a support is important to have sufficient function and some degree of mechanical strength as a place to seed and culture the cells in order to deliver the cells to organs such as the heart. That is, it is necessary to stably retain and engraft the cells in a uniform distribution state during culture, and to ensure good proliferation viability, and to be able to fix the suture or the like upon transplantation to the affected area after culture. In addition, it is necessary to use a support having a mechanical strength that withstands the compression caused by the beat of the heart.
  • MI myocardial infaction
  • the scaffolds used in this study are single-layered membranes that do not have pores for open structures, and there are problems with cell viability using two-dimensional matrix scaffolds and surgical methods for attachment to organs in the body. There is a problem in handling such as tearing when using.
  • a heart such as a high density of cells to be implanted because the pores of the support and the porosity should be more than 90%, there was a problem in applying.
  • the present inventors manufactured a patch having a constant thickness and porosity by using a three-dimensional support in the form of entangled biodegradable polymer fibers, which are spaced from each other, instead of implanting a two-dimensional cell culture product, which is a conventional method.
  • a three-dimensional support in the form of entangled biodegradable polymer fibers, which are spaced from each other, instead of implanting a two-dimensional cell culture product, which is a conventional method.
  • An object of the present invention is to provide a high success rate of delivery of cells, drugs, or bioactive substances to damaged tissues for the treatment of damaged tissues, and reversibly one-dimensional, two-dimensional, Or a three-dimensional expansion or contraction is excellent elasticity, the tissue regeneration patch to increase the angiogenesis and promote tissue regeneration of damaged tissue by moving cells, drugs, bioactive substances or a combination thereof to the ischemic site To provide.
  • Still another object of the present invention is to provide a method for producing the tissue regeneration patch.
  • the present invention is a three-dimensional support in the form of fluffy entangled with each other biodegradable polymer fibers (fluffy); And cells, drugs, bioactive substances, or a combination thereof contained on the surface or inside of the support.
  • fluffy biodegradable polymer fibers
  • the present invention (a) the step of preparing a spinning solution by dissolving the biodegradable polymer alone or mixed in an organic solvent; (b) spinning the spinning solution using a spinning machine and simultaneously volatilizing the organic solvent to prepare a support in which biodegradable polymer fibers are separated from each other and entangled with each other; (c) applying one or more bidirectional physical forces to the prepared support to inflate pores between the entire volume and intertwined polymeric fibers to provide a downy support; (d) providing a method for preparing a tissue regeneration patch, comprising introducing a cell, a drug, a bioactive substance or a combination thereof into the support.
  • the support included in the patch of the present invention can apply a physical force in one or more bidirectional to swell the voids between the total volume and the intertwined polymer fibers.
  • the patch of the present invention when the tissue regeneration patch of the present invention is implanted in a tissue such as the heart, the patch of the present invention has a mechanical strength to withstand the compression according to the heart beat (beating), while the patch according to the contraction and expansion of the heart In other words, it can be seen that it has elasticity that can be adjusted in volume or thickness depending on the environment in which the support is implanted, which can contract or expand interlockingly.
  • the patch for tissue regeneration of the present invention has a fluffy form that can inflate pores between the entire volume and intertwined polymer fibers by applying a physical force, so that the cells seeded in the patch are not pressed, proliferating and ischemic sites It is possible to secure a space to move to, and the diameter of the pores can be expanded to ensure oxygen permeability.
  • stem cells contained in the patch proliferate while living in the patch for a long time, myocardial Moved inward and confirmed to differentiate into cardiomyocytes.
  • blood vessels were generated in the ischemic site and the transplanted tissue, the thickness of the anterior wall of the left ventricle was increased, and superior myocardial regeneration was observed.
  • the stem cells when the stem cells are transplanted using the three-dimensional porous support of the present invention, the stem cells are directly transplanted to the damaged area and compared with the case where the stem cells are transplanted using the fibrin gel. It was confirmed that the survival rate and maintenance rate of the cells were significantly higher.
  • the gene can be stably loaded into the three-dimensional porous support of the present invention, it was confirmed that the controlled release of the gene locally.
  • the patch for tissue regeneration of the present invention can be attached directly to the target tissue, solving the problem of the low survival rate of the injected cells and the number of cells affecting myocardial tissue regeneration due to the specificity of the heart, a high rate at the damaged site
  • stem cells may be engrafted and maintained, and may be usefully used to promote tissue regeneration by increasing blood vessel generation at damaged tissue sites.
  • the present invention provides a patch for tissue regeneration.
  • Patch for tissue regeneration of the present invention is a three-dimensional support in the form of a fluffy (fluffy) entangled with the biodegradable polymer fibers spaced apart from each other; And it is characterized in that it comprises a cell, a drug, a bioactive material or a combination thereof contained on the surface or the inside of the support.
  • the three-dimensional support may be separated from each other without the polymer fibers adhered to each other, the polymer fibers are entangled with each other to form a gap between the fibers, the fibers have a two-dimensional fluffy form does not adhere to each other.
  • the support may be manufactured in a fluffy form by applying physical force in one or more directions to inflate pores between the entire volume of the support and the intertwined polymer fibers.
  • the biodegradable polymer fibers that are spaced from each other expand or contract reversibly in one, two, or three dimensions in conjunction with the expansion or contraction of the attached tissue.
  • the support of the present invention may be reversibly expanded or contracted depending on the heartbeat or the shape of the heart.
  • the total volume of the support and the pore size between the intertwined polymer fibers can be controlled.
  • the patch according to the present invention has excellent adhesion to the damaged site as a form of porous three-dimensional support
  • due to the excellent elasticity of the support it is possible to attach directly to the curved surface of the shape or organ to the tissue to be implanted.
  • the patch of the present invention may be appropriately set in thickness in consideration of oxygen permeability, securing of cell proliferation and movement space, and specificity of tissue, and manufactured in a size necessary to protect the affected part. Can be.
  • the thickness of the patch is preferably 50 ⁇ m or more and 1.5 cm or less, and when applied to the heart, preferably 0.5 to 3.5 mm thick, more preferably 1 to 3 mm thick.
  • the thickness of the patch is preferably controlled by expanding the total volume by applying physical force in one or more bidirectional directions during manufacture. It is desirable to expand the total volume by more than two times by applying a physical force to the initially produced support through the spinner. More preferably, the total volume is expanded 2 to 15 times.
  • the thickness of the patch in the case of a support that is inflated too thick due to physical expansion, a portion where cells do not enter occurs, and it is preferable to adjust the thickness of the patch according to the characteristics of the tissue to be transplanted.
  • the size and distribution of the pores of the support constituting the patch is a very important factor in determining cell growth, and the nutrient solution penetrates evenly into the support so that the cells grow well in an inter-connecting structure. It is preferable that the diameter of the said patch is 50 micrometers or more and 300 micrometers or less, and it is most preferable that it is 100 micrometers. It is a structure that helps the cell to grow to a size that can easily infiltrate the cells, discharge nutrients and wastes, and can easily grow blood vessels after transplantation, one or more bidirectional physical forces for expanding the diameter of the pores Can be added. In addition, due to the expansion of the pores, the support of the present invention can have a porosity of 30 to 90%, preferably have a porosity of 50 to 90% to maintain the three-dimensional support to grow the cells for tissue regeneration Can improve.
  • the shape and area of the support there is no particular limitation on the shape and area of the support, and it can be produced with a sufficient size to cover the damaged site.
  • the three-dimensional support of the present invention may have an orientation.
  • Cardiac muscles are directional and must withstand pressure, requiring a patch in the form of a network oriented in a constant direction.
  • the support was prepared by electrospinning, instead of the collector of the general stainless steel plate, a cylindrical drum collector was replaced, and a rotational speed of 1000 rpm or more was prepared in the form of a network-oriented patch.
  • the porous fibrous three-dimensional support having an orientation and the stem cell or / and growth factor drug are included therein.
  • the proliferation is improved while the cells have the orientation, and the shape of the myocardial fiber is similar to that of the original shape. Proliferation in the form was confirmed. In addition, growth factors are evenly distributed in the fiber, it was confirmed that the drug release is possible.
  • the patch for regenerating damaged tissue of the present invention is capable of regenerating tissue by attaching directly to most organs in which damaged tissue is required for cell delivery.
  • the damaged tissue may be attached to the heart, liver, skin, bone, nerve, or pancreas in which the damaged tissue is present to regenerate the damaged tissue, but is not limited thereto.
  • the cardiac stem cells are differentiated into cardiomyocytes, and the transplanted cells can regenerate damaged tissues by moving to surrounding cardiac tissues such as the peripheral myocardial infarction where the cardiomyocytes are damaged .
  • the cells contained in the support are preferably stem cells.
  • adipose derived stem cells As adult stem cells, adipose derived stem cells, umbilical cord blood derived stem cells, bone marrow derived stem cells, and the like can be used. In particular, for the regeneration of damaged cardiac tissue, it is preferable to use cardiac stem cells that are well differentiated into cardiac cells and have a cardiac regeneration effect and are able to move into heart tissue.
  • the present invention uses the myocardial stem cells of the mouse heart, but is not limited thereto, and similarly to the stem cells collected from all kinds of mammalian species including humans, rats, mice, monkeys, and the like. Apply. When a patch containing stem cells is transplanted into damaged tissue, it can be confirmed that the stem cells are differentiated into cells of the tissue.
  • cardiac stem cells are used to differentiate using cardiac specific markers.
  • markers include, but are not limited to, myosin heavy chain (MHC), cardiac troponin I (cTnI), cardiac troponin T (cTnT), ⁇ -cardiac actin, ⁇ -actinin and myosin light chain (MLC2). Phenotypes of these cells can be assessed by rhythmic contractions and expression of markers associated with the heart.
  • the drug in the patch for damaged tissue regeneration of the present invention, can be applied in the patch for rapid recovery of the damaged tissue.
  • the drug may use a gene involved in tissue regeneration, a gene that improves the performance of stem cells, or a regenerative protein.
  • the regenerative protein is a vascular endothelial growth factor (VEGF) -A, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, neurophylline, (FGF) -1, FGF-2 ( bFGF), FGF-3, FGF-4, FGF-5, FGF-6, Angiopoietin 1, Angiopoietin 2, Erythropoietin, BMP-2, BMP-4, BMP-7, TGF It is preferably one or more selected from the group consisting of -beta, IGF-1, osteopontin, playotropin, activin, and endorumin-1, but is not necessarily limited thereto.
  • Vascular endothelial growth factors may act independently or in combination with each other, where the angiogenesis factors act synergistically and are more effective than the sum of the effects of separate individual factors.
  • VEGF which is a vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • the drug can be included in the support to deliver the drug, thereby maximizing the tissue regeneration effect, and in particular, by slowly releasing the regeneration period, the effect can be enhanced.
  • the cells or stem cells can be included on the surface or / and the support.
  • the three-dimensional support of the biodegradable polymer material is preferably made of a synthetic polymer that can be decomposed by hydrolysis in terms of processability, sterility, and infectivity, in particular ⁇ and ⁇ - It is preferably made of a hydrolyzable polymer of hydroxycarboxylic acid.
  • the biodegradable polymer is polylactic acid (PLA), polyl-lactic acid (PLLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L-lactide- co-glycolide); PLGA), biodegradable aliphatic polyesters such as poly (caprolactone), diol / diacid based aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydr Oxybutyrate) and poly (hydroxy valerate), it is preferable to use at least one synthetic polymer selected from the group consisting of polylactic acid (PLA) having a molecular weight of 100,000 to 350,000 kD, Most preferably, polyl-lactic acid (PLLA) is used, but is not necessarily limited thereto.
  • PLA polylactic acid
  • PLLA polyl-lactic acid
  • PGA polyglycolic acid
  • PGA poly (D, L-lactic acid-co-glycoli
  • the present invention provides a method for producing a patch for tissue regeneration.
  • any organic solvent used to prepare the spinning solution as a polymer solution can be used as long as it is a volatile organic solvent having a low boiling point, chloroform, dichloromethane, dimethylformamide, dioxane , Acetone, tetrahydrofuran, trifluoroacetic, 1,1,1,3,3,3, -hexafluoroisopropyl propanol or combinations thereof are preferred, in particular with high volatile dichloromethane and solubility Very low acetone cosolvents are suitable.
  • Step (b) is to prepare a support using the spinning solution using a spinning machine.
  • the radiator may be an electrospinner, but is not limited thereto.
  • the spinning process may be as follows. After a constant current flows in the voltage generator, an electric field is formed between the nozzle and the collector, and the polymer solution filled in the spinning liquid reservoir is spun by the force of the electric field and the pressure of the pump.
  • the condition of the electrospinning machine is preferably 10 to 20 cm in spinning distance, voltage of 10 to 20 kv, discharge rate of 0.05 to 0.15 ml / min, but is not limited thereto.
  • the polymer fibers are separated from each other and entangled with each other to form fibers having various diameters of semi-micro to nano size. And the spun form contains suitable pores in the form of intertwined fibers.
  • Step (c) is a step of providing a support in the form of a fluffy by applying a physical force in one or more directions in both directions to the support prepared by using the spinning machine to inflate pores between the total volume and intertwined polymer fibers.
  • the support has a fluffy form, the thickness of which extends from 50 ⁇ m to 1.5 cm. It is also desirable to expand at least about two times the volume of the original support produced by the spinning method. More preferably, the volume is expanded about 2 to 15 times.
  • the step (d) is a step of introducing a cell, a drug, a bioactive substance or a combination thereof into the support of the step (c) is a cell that can be used as stem cells capable of inducing tissue regeneration, cells capable of differentiation Drugs include genes involved in tissue regeneration, genes that improve stem cell performance, and regenerative proteins.
  • the drug solution patch can be prepared by mixing the drug solution of the aqueous phase with the emulsion method and laminating the mixed solution by spinning.
  • the drug dispersed in the biodegradable polymer solution in the emulsion step according to the invention is emulsified in a therapeutically effective amount in the biodegradable polymer solution using a surfactant known in the art, for example, span 80, uniform in the form of water-in-oil emulsion. Can be distributed.
  • the drug-seeded patch is implanted into the damaged tissue and exposed to the aqueous environment to release the protein it contains. At the beginning of the release, it is diffused and released from the patch, and when the decomposition of the support itself starts, the amount of release from the lost gap increases. Release rates and modalities can be varied depending on protein concentration, flow / water ratio, and application area.
  • the patch of the present invention prepared by the radiation method is excellent in cell retention and viability, and furthermore, by culturing cells or progenitor cells derived from tissues in an artificial environment and / or in vivo using this support, It is possible to prevent the infiltration of inflammatory cells at the time and to produce a three-dimensional cell assembly having properties close to the original tissues of the living body that are compatible with the surrounding tissues.
  • Tissue regeneration patch according to the present invention is easier to handle (handling) compared to the injection method, single cell (single cell) and the method of applying the cells in the form of a two-dimensional support, cell survival after transplantation is high and requires cell transplantation It is possible to enhance the therapeutic efficacy of cell therapies at the disease site.
  • the patch for tissue regeneration according to the present invention has elasticity and flexibility that can be expanded or reduced in volume or thickness depending on the environment in which the biodegradable polymer fibers spaced apart from each other entangled with each other, depending on the environment to be implanted, The size and thickness of the pores can be inflated so that the seeded cells are not pressed, sufficient space for the cells to proliferate and move, and oxygen permeability is secured to promote cell proliferation, movement, and blood vessel formation.
  • the cell delivery success rate is increased and the cell ischemic site By moving to, it increases the angiogenesis of damaged tissue and has the effect of promoting tissue regeneration.
  • Example 1 is a (a) photorealistic image and (b) differential scanning micrograph of the PLLA fibrous porous three-dimensional support patch prepared in Example 1.
  • Figure 2 is a photograph showing that the thickness of the support can be inflated by more than 1 cm in a state in which the volume per void and the amount of the support is widened by applying a physical force to the support prepared by electrospinning.
  • FIG. 3 shows the reversible elasticity of the support by gradually increasing the volume per void and the amount of the support by applying a physical force to the support prepared through electrospinning.
  • the rightmost support is a support of the two-dimensional membrane structure, showing a significant difference in the porosity, porosity between the three-dimensional support and the structure and fibers of the present invention.
  • FIG. 4 is a differential scanning micrograph of a PLLA fibrous porous three-dimensional support patch prepared to have an orientation.
  • Example 5 is a differential scanning microscope photograph in which cardiac stem cells are attached to randomly radiated non-oriented patches prepared according to Example 1, and (b) cardiac stem cells to patches having an orientation prepared according to Example 2. Is a differential scanning microscope picture attached.
  • Figure 6 is a cell grafted in a fibrous porous three-dimensional support patch having an orientation, and after 48 hours H & E staining (a) a cross-sectional microscopic observation of the cross section, (b) a cross-sectional microscopic photograph.
  • Figure 7 shows the results of confirming the distribution of the growth factor in the orientation-oriented three-dimensional support containing the growth factor of Example 3 through a fluorescent reagent.
  • FIG. 8 is a graph showing the cumulative amount of VEGF drug released from the oriented three-dimensional support containing the growth factor of Example 3.
  • Figure 9 shows the number of cells measured on day 1, day 4, day 7, and 14 according to Experiment 3.
  • Figure 10 shows the growth of cells seeded in the three-dimensional support of Example 1 and the three-dimensional support having the orientation of Example 2.
  • Figure 2 shows the proliferation of cells in the non-directional three-dimensional scaffold of Example 1
  • Figure 4 shows the growth of cells in the three-dimensional scaffold with orientation.
  • FIG. 11 is a H & E, ⁇ SA, MHC, and TnT immunostaining photograph of the stem seeded with cardiac stem cells according to Experimental Example 4 and cultured for 2 weeks in vitro in (a) growth medium composition and (b) differentiation medium composition.
  • FIG. 12 (a) and 12 (b) are micrographs obtained by H & E staining of tissues after 4 days of implanting a patch seeded with heart stem cells on the outer pericardial surface of normal heart tissues according to Experimental Example 5.
  • FIG. 12 (a) and 12 (b) are micrographs obtained by H & E staining of tissues after 4 days of implanting a patch seeded with heart stem cells on the outer pericardial surface of normal heart tissues according to Experimental Example 5.
  • Figure 13 is a (a) picture immediately after implanting the patch without seeding the stem cells of Comparative Example 1 in the myocardial infarction, (b) tissue photograph after 14 days, (c) heart stem of Example 1 in the myocardial infarction Pictures immediately after transplanting the seed seeded patch and (d) 14 days later tissue.
  • FIG. 14 is a photograph obtained by H & E staining of tissue obtained after 14 days according to Experimental Example 7, (a) photografted with patch of Example 1 seeded with cardiac stem cells, and (b) comparative example without cardiac stem cells This is a photo of a patch of 1.
  • FIG. 15 is a tissue photograph obtained after 14 days in accordance with Experimental Example (a) immunostained tissue photograph, (b) DAPI stained nucleus, and (c) (a) and (b).
  • FIG. 16 is a tissue photograph obtained after 14 days according to Experimental Example (a) TnT immunostained tissue photograph, (b) DAPI stained nuclei, and (c) (a) and (b) superimposed.
  • Figure 17 is a (a) SMA immunostained small artery / vein image, (b) DAPI stained nuclei, (c) (a) and (b) of the tissue obtained after 14 days according to Experimental Example 7 It is a photograph.
  • FIG. 19 is a low-magnification photograph of the patch and heart tissue 14 days after transplantation according to Experimental Example 7, (a) transplanted DiI-labeled heart stem cells in red fluorescence, (b) DAPI stained nucleus, (c) It is the photograph which superimposed (a) and (b).
  • Figure 20 is a high magnification micrograph of the myocardial tissue showing the location of the cardiac stem cells transplanted in the myocardial layer of myocardial infarction rat, the upper part is attached patch and the lower part shows the inside of the myocardium, the red fluorescence of (a) is DiI-labeled cardiac stem cells are shown, blue fluorescence in (b) represents nuclei labeled with DAPI, and (c) is a photograph of (a) and (b) superimposed.
  • FIG. 21 is a photograph obtained by H & E staining of tissue obtained after 28 days according to Experimental Example 8, (a) photografted with patch of Example 1 seeded with cardiac stem cells, and (b) Comparative Example 1 without cardiac stem cells This is a photo of a patch of.
  • FIG. 22 shows (a) fibrosis area measurement results when the patch of Example 1 is implanted and the patch of Comparative Example 1 without stem cells is transplanted as a histological analysis of tissue obtained after 28 days according to Experimental Example 8, (b) Heart thickness measurement results.
  • FIG. 23 shows the results of cell migration and infiltration in the case where the body is inflated too thick by additional physical extension to the three-dimensional support.
  • the figure on the left shows a case where the cell proliferation has been successfully made by inflating to an appropriate thickness, and the figure on the right shows that a part (white) where cells cannot enter the inside of the support has occurred.
  • FIG. 24 shows that the support, the growth factor-containing support, the growth factor, and the support containing the heart stem cells were transplanted into myocardial tissue, and then the regeneration effect to the myocardium was compared through tissue staining.
  • FIG. 25 shows that angiogenesis is increased after implantation of a scaffold, a scaffold containing a growth factor, a scaffold containing a growth factor, and a cardiac stem cell into myocardial tissue, by SMA positive vessel formation analysis. It is.
  • FIG. 26 shows the PLLA fibrous porous three-dimensional support (PLLA) of Example 2, seeded with cardiac stem cells, and the PLLA fibrous porous three-dimensional support (PLLA / VEGF) of Example 2, seeded with cardiac stem cells and VEGF.
  • Scanning electron micrograph. (a) and (d) are photographs of one day after incubation, (b) and (e) are five days after incubation, and (c) and (f) are seven days after incubation.
  • FIG. 27 shows the PLLA fibrous porous three-dimensional scaffold of Example 2 seeded with cardiac stem cells and the PLLA fibrous porous three-dimensional scaffold of Example 2 seeded with cardiac stem cells and VEGF, 1, 5, and 7 It shows the result of confirming the degree of proliferation in the day.
  • FIG. 28 shows a patch of Example 2 (PLLA) without cardiac stem cells and growth factors seeded, a PLGF loaded PLLA fibrous porous three-dimensional support patch (PLLA / VEGF) and cardiac stem cells and VEGF.
  • PLLA PLLA
  • VEGF PLLA fibrous porous three-dimensional support patch
  • FIG. 28 shows a patch of Example 2 (PLLA) without cardiac stem cells and growth factors seeded, a PLGF loaded PLLA fibrous porous three-dimensional support patch (PLLA / VEGF) and cardiac stem cells and VEGF.
  • A Fibrosis area measurement result
  • b Heart thickness measurement result when the PLLA fibrous porous three-dimensional support patch (PLLA / VEGF / rCSC) of Example 2 implanted was implanted.
  • Example 29 shows a patch of Example 2 (PLLA) without cardiac stem cells and growth factors seeded, a VEGF loaded PLLA fibrous porous three-dimensional support patch of Example 2 (PLLA / VEGF) and cardiac stem cells and VEGF (A) EF (ejection fraction), and (b) fractional shortening when the PLLA fibrous porous three-dimensional support patch (PLLA / VEGF / rCSC) of Example 2 was seeded was measured. .
  • FIG. 30 shows cell viability at the transplanted tissue site of the group implanted with stem cells directly to the damaged heart, the group transplanted with fibrin gels containing stem cells, and the group implanted with the three-dimensional scaffold of the present invention containing stem cells; It shows retention rate.
  • FIG. 31 is a SEM photograph showing loading of a plasmid DNA (pDNA) for gene transfer into a three-dimensional support of the present invention having an orientation.
  • FIG. (a) SEM image of the three-dimensional scaffold of the present invention having an orientation, (b) a patch coated with a pDNA complex for transfection, (c) a SEM image of a rat heart stem cell-attached patch 24 hours after seeding, (d) An image of the surface of the scaffold observed with a fluorescence microscope.
  • FIG. 32 is a confocal image of human VEGF expression observed in rat heart stem cells after transfection of plasmid DNA.
  • Fig. 34 is a diagram showing the expression level of the hVEGF gene transfected into rat heart stem cells into subsequent proteins.
  • PLLA / PLGA patch control seeded with cardiac stem cells
  • PLLA / PLGA patch Bolus delivery seeded with heart stem cells transfected with pVEGF complex
  • PLLA / PLGA / VEGF patch seeded with heart stem cells
  • Example 1 Preparation of a fibrous porous three-dimensional support patch containing stem cells 1
  • Polyl-lactic acid (PLLA) was dissolved in a solvent of dichloromethane / acetone (the volume of acetone was 10% to 40% of the total solution, the exact volume tested 20%), and the concentration of the polymer was 14 to 20% (the exact concentration tested). 15%) to prepare a solution.
  • DH High Voltage Generator model name CPS-40KO3VIT
  • electrospinner manufactured by Chungpa EMT Co., Ltd. (Korea)
  • electrospinning was performed under conditions of solution discharge rate of 0.06 ml / min, voltage of 10 kv, and electric field of 15 cm. It was.
  • Electrospinning is performed under conditions of 15-25 ° C and 10-40% humidity so that the solvent is volatilized before the fibers are accumulated on the stainless steel plate collector, so that the biodegradable polymer fibers can be separated from each other and entangled with each other.
  • the fiber thickness of the support was about 7 ⁇ m in diameter and the thickness of the support was about 300 ⁇ m.
  • the fleece support prepared by applying physical force to the support had a pore diameter of 50-300 ⁇ m, a porosity of 50-90%, and a thickness of 1 mm or more (FIG. 1).
  • the thickness of the support was measured after inflating the voids between the fibers and the total volume of the support by applying a physical force in one or more directions in the electrospinning support. As a result, it was confirmed that the thickness of the tissue regeneration patch of the present invention can be extended to 1 cm or more (FIG. 2). Others could be identified ( Figure 3).
  • the scaffolds of the present invention can be separated from each other between the fibers, due to the characteristics of the fluffy form it can be seen that the reversible expansion or contraction in conjunction with the expansion and contraction of the proliferation and movement of the cells, or the attached tissue.
  • a patch containing a three-dimensional support, that is, a patch having an orientation was produced (FIG. 4).
  • vascular endothelial growth factor was seeded on the three-dimensional scaffold patches prepared in Examples 1 and 2 to prepare patches including a fibrous porous three-dimensional scaffold containing stem cells and growth factors.
  • Example 4 Preparation of fibrous porous three-dimensional support patches containing genes
  • PLGA was dissolved in acetone / ethanol mixed solvent to prepare a suspension, which was then emulsified with aqueous pVEGF / CPP complex or FITC, which was electrosprayed to coat the support surface of Example 2 and lyophilized. At this time, the amount of pVEGF complex was fixed at 3 ⁇ g per support (8 * 8).
  • Unattached cells were washed off and fixed for 20 minutes with 2.5% glutaraldehyde solution. After fixation, the samples were dehydrated sequentially with 70%, 80%, 90% and 100% ethanol for 10 minutes each and thoroughly vacuum dried. The sample was then coated with gold.
  • FIG. 5 (b) The result of observing this with a differential scanning microscope is shown in FIG. It can be seen that the cells adhered well to the prepared support, and for the support having the alignment of Example 2, the alignment of the cells was confirmed along the fibers having the alignment (FIG. 5 (b)).
  • Example 2 After stabilizing cell adhesion for 48 hours to confirm that cells were seeded at high density, the three-dimensional scaffold-cell aggregates obtained in Example 2 were washed with buffer and fixed with 3.7% formaldehyde solution for one day. 4 ⁇ m thick slides were prepared after paraffin pome and observed under a microscope by staining with H & E (Hematoxylin and Eosin) (FIG. 6).
  • the cells are seeded at a high density in the photograph of the transverse section (FIG. 6A), and in the photograph of the cross section, the cells are present at a high density as well as outside (FIG. 6 (FIG. 6A). b)). This is because the patch for tissue regeneration of the present invention expands the size of the pores by physical expansion, and it is easy for the stem cells to penetrate into the support because the size of the pores of the support is large.
  • the growth factor and release rate of the growth factor in the support including the growth factor was measured.
  • the patch of the present invention including a growth factor was found to be able to sustain local and sustained drug release in the myocardial infarction site.
  • the patch containing the stem cells and growth factors of the present invention was found to be effective in tissue regeneration by continuously introducing growth factors into the stem cells by continuously releasing growth factors.
  • aSA ⁇ -sarcomeric actinin
  • MHC myosin heavy chain
  • TnT troponin-T
  • the degree of differentiation was high in the patch cultured in the differentiation medium, and the differentiation into cardiomyocytes was also observed in the culture medium in the growth medium. This is believed to be the result of using stem cells in the heart.
  • Sprague-Dawley rats (8-9 weeks) were anesthetized with Isofurane. Anesthetized rats maintained breathing under ventilated under positive pressure.
  • Example 1 In order to observe the stability of the patch using a porous three-dimensional scaffold seeded with stem cells, the patch prepared in Example 1 was implanted on the epicardium surface of normal heart tissue and evaluated 4 days later. The degree of fusion with the tissue and the degree of inflammatory response after H & E staining are shown in FIG. 12.
  • a portion circled in the region P (Periphery) of FIG. 12 represents blood vessels, which shows that the growth of blood vessels was made into the patch. In other words, you can see that the fusion of the patch and the heart tissue is made well. In addition, since tissue formation is observed at the interface (region I of FIG. 12) and the periphery (region P of FIG. 12) between normal heart tissue and the patch with respect to cell differentiation, it can be observed that the cells also fuse with cardiomyocytes.
  • MI model Preparation of myocardial infarction
  • Sprague-Dawley rats (8-9 weeks) were anesthetized with Isofurane. Anesthetized rats maintained breathing under ventilated under positive pressure. Thoracotomy was performed between the second and third left ribs. After the pericardium was cut out and the heart was pushed out by the pressure of the rib cage, the left coronary artery was ligated with 7-0 plastic prolene and ligated to induce myocardial infarction. After performing left anterior Descending (LAD) ligation and confirming the generation of ischemic sites, the patch seeded with stem cells or stem cells and growth factors of Examples 1 to 3 of size 10x10 mm was immediately obtained. -0 silk was attached to the infarcted myocardium zone and the infarction border zone, respectively, of the epicardium surface.
  • LAD left anterior Descending
  • the disease model was prepared in the same manner as in (1) except that the patches of Examples 1 to 3 were implanted into the anterior wall of the left ventricle and sacrificed 28 days.
  • Example 1 (2) Immediately after the wound, the patch of Example 1 and the patch of Example 1, which were not seeded with the stem cells (Comparative Example 1), were implanted into the anterior wall of the left ventricle. Sacrifice and cardiac tissue were observed by paraffin sections fixed with 10% buffered formaldehyde.
  • Figure 13 shows a picture immediately after implanting the patch seeded with the cardiac stem cells of Example 1 in the myocardial infarction portion
  • (b) shows a tissue picture after 14 days.
  • (c) shows a photograph immediately after implanting the patch without seeding the stem cells of Comparative Example 1 in the myocardial infarction, and (d) shows the histological image after 14 days.
  • the prepared tissue was prepared as a slide and stained with H & E (hematoxylin and eosin) (Fig. 14). After 14 days of coronary artery ligation, myocardial infarction was successfully induced, and myocardial infarction and cardiac dilatation were observed.
  • H & E hematoxylin and eosin
  • the patches of Example 1 and Comparative Example 1 were found to be completely fused with the LV anterior wall.
  • the shape of the implanted patch is well maintained, and the LV anterior wall thickness of the heart is a cellless patch. It can be seen that the thicker than the heart of Comparative Example 1 implanted.
  • the heart implanted with the patch of Example 1 had less LV dilatation, and myocardial regeneration (white arrow area) was prominently observed.
  • the tissue transplanted with the seeded patch of cardiac stem cells was stained with a myocardial specific antibody and stained with a fluorescein isothiocyanate-conjugated secondary antibody. It was. They represent ⁇ -sarcomeric actinin (aSA) and troponin-T (TnT) cardiomyocyte markers, which are sarcomere proteins involved in cardiomyocyte contraction.
  • aSA ⁇ -sarcomeric actinin
  • TnT troponin-T
  • Figure 15 shows an aSA immunostained photograph and (b) shows all nuclei labeled with DAPI. 15 (c) shows that stem cells in the patch differentiated into aSA cardiomyocytes 14 days after the patch was implanted into the tissue as a result of overlapping (a) and (b).
  • FIG. 16 (a) shows a TnT immunostained photograph and (b) shows all nuclei labeled with DAPI. 16 (c) shows that stem cells in the patch differentiated into TnT cardiomyocytes 14 days after the patch was transplanted into the tissue as a result of overlapping (a) and (b).
  • Figure 17 (a) is a SMA immunostained arterial / vein photo and (b) shows all nuclei labeled with DAPI. 17 (c) shows that a small artery / vein grows in the patch as a result of overlapping (a) and (b).
  • FIG. 18 shows CD34 immunostaining of capillaries and (b) shows all nuclei labeled with DAPI. 18 (c) shows that capillaries grow in the patch as a transplant tissue as a result of overlapping (a) and (b).
  • FIG. 19 is a low-magnification photograph of the patch and heart tissue 14 days after transplantation.
  • FIG. 19 (a) is a photograph showing red fluorescence of transplanted DiI-labeled heart stem cells and (b) is a DAPI stained image of the nucleus.
  • (c) is a photograph in which (a) and (b) are superimposed, and a cell aggregate that is DiI positive in the patch is observed. This confirms that stem cells survive in long-term patches and migrate into the myocardium.
  • Figure 20 is a high magnification micrograph of the myocardial tissue showing the location of the heart stem cells transplanted in the myocardial layer of myocardial infarction rat 14 days after transplantation, the upper part is the patch portion attached to the inner side of the myocardium toward the bottom.
  • Red fluorescence in (a) represents DiI-labeled heart stem cells
  • blue fluorescence in (b) represents nuclei labeled with DAPI
  • (c) is a photograph of (a) and (b) superimposed, with DiI Labeled stem cells were also found in the myocardial layer and myocardial infarction areas lacking myocardial cells, confirming that the transplanted cells migrate to the surrounding heart tissue.
  • the tissue obtained from the animal model of Experimental Example 6 (2) was prepared as a slide and stained with H & E (hematoxylin and eosin) (Fig. 19). After 28 days of coronary artery ligation, myocardial infarction was successfully induced, and myocardial infarction and cardiac dilatation were observed.
  • H & E hematoxylin and eosin
  • the implanted patch of the heart stem cells seeded in Example 1 was well maintained, and the LV anterior wall thickness of the heart was compared with the transplanted patch without cells. You can see that it is thicker than the heart of Example 1.
  • the heart implanted with the patch of Example 1 had less LV dilatation, and regeneration (arrow area) of myocardium was prominently observed (FIG. 21).
  • the fibrosis area and the heart thickness of the tissues to which the patches of Example 1 and Comparative Example 1 were implanted were measured 28 days after the patch implantation.
  • the patch seeded cardiac stem cells of Example 1 it can be seen that the fibrotic area is significantly reduced (Fig. 22 (a)).
  • LV anterior wall thickness was confirmed to be thicker than the heart of Comparative Example 1 in which the cell-free patch was implanted (FIG. 22). (b)).
  • Example 2 An additional physical force was applied to the support of Example 2 to prepare a support having a density of about 5 times reduced. Specifically, the cell migration effect of the patch according to Example 2 and the support having a density reduced by about 5 times from 14.5 g / cm 3 to 2.7 g / cm 3 was confirmed.
  • the stem cell seeded patch was implanted into the damaged heart of the animal model of Experimental Example 4. Observing cardiac tissue on day 14 after implantation revealed that one or more bidirectional physical forces exerted on the support to inflate pores between the volume of the support and the intertwined polymer fibers resulted in migration and penetration of the cells into and within the support. It could be confirmed that the increase (Fig. 23). However, when stem cells were seeded on the overblown scaffold by applying physical expansion, the cells could not migrate into the scaffold. FIG. 23 shows that a cell (white) where cells cannot enter the support and tissues is generated when a patch that inflates the support excessively by applying a physical force (Fig. 23, right) is implanted.
  • Myocardial regeneration of the patch containing the growth factor in the fibrous porous support according to Example 3 was confirmed. Specifically, the group implanted with only the fibrous porous three-dimensional support in the damaged myocardial tissue portion, the group implanted with a patch containing the growth factor in the fibrous porous three-dimensional support, the stem cells and the growth factor in the fibrous porous three-dimensional support The patch was divided into groups to be transplanted, and tissue staining was performed to determine whether the myocardium was regenerated and blood vessels were generated.
  • the patch comprising the three-dimensional support of Example 2, and the patch containing the support loaded with VEGF on the three-dimensional support of Example 2 on the day corresponding to 1, 5 and 7 days while incubated under the growth medium composition for 7 days Its morphological characteristics were observed and the results are shown in FIG.
  • the fibrosis area and heart thickness of the tissues were measured 28 days after patch implantation.
  • the control factor growth factor and the group transplanted with the patch of Example 2 without seeding the stem cells PLLA
  • the patch loaded with VEGF on the three-dimensional support of Example 2 without the seeding of the stem cells PLLA / VEGF
  • the effect was confirmed in the group transplanted and the group implanted with the patch of Example 2 (PLLA / VEGF / rCSCs) seeded with VEGF and cardiac stem cells, and the results are shown in FIG. 29.
  • both the PLLA / VEGF and PLLA / VEGF / rCSCs group was less fibrous area than the control group, it was confirmed that the thickness of the left ventricle wall thickness was also thicker than the control group.
  • the patch containing both VEGF and cardiac stem cells showed a 10% smaller fibrosis area than the control group, and about 1.5 times thicker in the left ventricular wall thickness.
  • the control group was transplanted with the patch of Example 2 without growth factor and stem cells seeded (PLLA), and the patch (PLLA / VEGF) with VEGF loaded into the three-dimensional support of Example 2 without stem cells seeded was transplanted.
  • Left ventricular ejection fraction (EF) and fractional shortening (FS) of the group and the group implanted with the patch of Example 2 (PLLA / VEGF / rCSCs) seeded with VEGF and cardiac stem cells were measured. It was confirmed whether the cardiac function of the group implanted with the patch of the present invention was improved.
  • a group directly injecting heart stem cells into the damaged myocardium region of the animal model, a group implanted with a fibrin gel containing heart stem cells, a heart containing heart stem cells in a fibrous porous three-dimensional support according to the present invention The patch was divided into groups transplanted to check the viability of the transplanted cardiac stem cells and the maintenance rate at the damaged myocardial tissue site.
  • the cell viability and maintenance effect of about 4 times compared to the group injected with stem cells directly, and about 9 times compared to the group transplanted by introducing stem cells into the fibrin gel. It could be confirmed that the increase (Fig. 30). From this, the tissue regeneration patch containing cells according to the present invention has high viability and maintenance effect at the damaged tissue site, and thus, cell delivery to the damaged tissue is very effective, which may result in superior tissue regeneration effect compared to other tissue regeneration methods. It can be seen that.
  • ELISA enzyme-linked immunosorbent assay
  • hVEGF165 was stably transfected into cardiac stem cells, indicating that they were expressed in the nucleus (top of FIG. 32).
  • 3 ⁇ g of the pVEGF loaded support was incubated for 14 days at a temperature of 1 mL of PBS (pH 7.4) at 37 ° C. All samples were stirred at constant speed (30 rpm). At defined time intervals (1 hour, 6 hours, 1 day, 2 days, 5 days, 7 days, 10 days and 14 days), the release medium was removed and freshly ground. The removed medium was centrifuged and then the amount of pDNA released from the support was determined using PicoGreen reagent as a fluorescent dye (invitrogen, Eugene, Oregon, USA). The ratio of released pDNA was calculated as the ratio of the mass of total pDNA loaded on the support to the mass of released pDNA.
  • the expression levels of the hVEGF protein of the Sustained release group and the group seeded with pVEGF transfected heart stem cells on the oriented PLLA / PLGA scaffold were compared with each other.
  • rat heart stem cells were injected with the same amount of pVEGF complex as the Sustained release group, and the expression level of hVEGF protein was analyzed by ELISA kit, and the results are shown in FIG. 34 and Table 1.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Organic Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Materials Engineering (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Preparation (AREA)
PCT/KR2013/005094 2005-08-26 2013-06-10 Patch destiné à la régénération tissulaire, comprenant un échafaudage tridimensionnel poreux fibreux Ceased WO2013183976A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/047,948 US20210308335A1 (en) 2005-08-26 2013-10-07 Fibrous 3-Dimensional Scaffold Via Electrospinning For Tissue Regeneration and Method For Preparing the Same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2012-0061818 2012-06-08
KR20120061818 2012-06-08

Related Child Applications (2)

Application Number Title Priority Date Filing Date
PCT/KR2006/003390 Continuation-In-Part WO2007024125A1 (fr) 2005-08-26 2006-08-28 Echafaudage fibreux tridimensionnel produit par filage electrostatique pour regeneration tissulaire et procede permettant de preparer cet echafaudage
US12/064,801 Continuation-In-Part US20080233162A1 (en) 2005-08-26 2006-08-28 Fibrous 3-Dimensional Scaffold Via Electrospinning For Tissue Regeneration and Method For Preparing the Same

Publications (1)

Publication Number Publication Date
WO2013183976A1 true WO2013183976A1 (fr) 2013-12-12

Family

ID=49712304

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2013/005094 Ceased WO2013183976A1 (fr) 2005-08-26 2013-06-10 Patch destiné à la régénération tissulaire, comprenant un échafaudage tridimensionnel poreux fibreux

Country Status (2)

Country Link
KR (1) KR101540845B1 (fr)
WO (1) WO2013183976A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108079378A (zh) * 2017-12-22 2018-05-29 中国科学院上海硅酸盐研究所 具有活性离子释放功能和纳米结构的有机/无机复合心肌补片材料及其制备方法
CN111714248A (zh) * 2020-05-08 2020-09-29 南开大学 一种促进细胞快速增殖及促进其细胞外基质沉积的血管支架及脱细胞基质人工血管
CN112210888A (zh) * 2020-10-23 2021-01-12 中原工学院 利于组织再生的聚乳酸弹性无纺材料及其制备方法

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10371610B2 (en) 2016-02-23 2019-08-06 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof
KR20170099738A (ko) 2016-02-23 2017-09-01 노을 주식회사 접촉식 염색 패치 및 그 제조 방법
WO2017146507A1 (fr) * 2016-02-23 2017-08-31 노을 주식회사 Patch de culture, procédé de culture, procédé et appareil d'analyse de culture, et procédé et appareil de test d'un médicament
KR102478639B1 (ko) 2016-02-23 2022-12-19 노을 주식회사 표지 물질을 저장하는 패치, 이를 이용하는 조직 진단 방법 및 장치
US10018615B2 (en) 2016-03-08 2018-07-10 Lawrence Livermore National Security, Llc Three-dimensional electronic scaffold for cardiac applications
KR20200073787A (ko) 2018-12-14 2020-06-24 글라드아이 주식회사 생체세포가 배양된 생체이식용 다공성 바이오 패치
CN110464880B (zh) * 2019-09-27 2021-10-26 中国医学科学院生物医学工程研究所 一种人工胰岛或人工胰腺及其制备方法
CN111785446A (zh) * 2020-08-11 2020-10-16 广东工业大学 一种二维材料基心脏起搏器连接线及其制备方法
CN114225106B (zh) * 2021-12-23 2023-03-21 广东工业大学 一种多孔纳米纤维生物膜及其制备方法和应用
KR102454631B1 (ko) * 2022-03-02 2022-10-14 이하민 줄기 세포 유래 성분을 포함하는 화장품 및 이의 제조 방법

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20050040186A (ko) * 2003-10-28 2005-05-03 이승진 생분해성 고분자로 이루어진 섬유형 다공성 지지체 및그의 제조방법
US6899873B2 (en) * 2001-02-02 2005-05-31 The Regents Of The University Of Michigan Method of forming micro-tubular polymeric materials
KR100496354B1 (ko) * 2002-03-27 2005-06-20 서울산업대학교 산학협력단 생분해성 고분자 지지체층을 포함하는 하이브리드인공혈관 및 그의 제조 방법

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100564366B1 (ko) * 2003-11-19 2006-04-07 재단법인서울대학교산학협력재단 나노 섬유형 부직포를 이용한 조직 재생용 차폐막 및 그의 제조 방법
KR100875189B1 (ko) * 2005-08-26 2008-12-19 이화여자대학교 산학협력단 전기방사를 이용한 조직 재생용 섬유형 삼차원 다공성 지지체 및 그의 제조방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6899873B2 (en) * 2001-02-02 2005-05-31 The Regents Of The University Of Michigan Method of forming micro-tubular polymeric materials
KR100496354B1 (ko) * 2002-03-27 2005-06-20 서울산업대학교 산학협력단 생분해성 고분자 지지체층을 포함하는 하이브리드인공혈관 및 그의 제조 방법
KR20050040186A (ko) * 2003-10-28 2005-05-03 이승진 생분해성 고분자로 이루어진 섬유형 다공성 지지체 및그의 제조방법

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHIM ET AL.: "Novel three-dimensional scaffolds of poly(L-lactic acid) microfibers using electrospinning and mechanical expansion: Fabrication and bone regeneration", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B: APPLIED BIOMATERIALS, vol. 95B, no. IS. 1, 19 August 2010 (2010-08-19), pages 150 - 160 *
YANG ET AL.: "Fabrication of nano-structured porous PLLA scaffold intended for nerve tissue engineering", BIOMATERIALS, vol. 25, no. 10, May 2004 (2004-05-01), pages 1891 - 1900 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108079378A (zh) * 2017-12-22 2018-05-29 中国科学院上海硅酸盐研究所 具有活性离子释放功能和纳米结构的有机/无机复合心肌补片材料及其制备方法
CN108079378B (zh) * 2017-12-22 2020-09-18 中国科学院上海硅酸盐研究所 具有活性离子释放功能和纳米结构的有机/无机复合心肌补片材料及其制备方法
CN111714248A (zh) * 2020-05-08 2020-09-29 南开大学 一种促进细胞快速增殖及促进其细胞外基质沉积的血管支架及脱细胞基质人工血管
CN112210888A (zh) * 2020-10-23 2021-01-12 中原工学院 利于组织再生的聚乳酸弹性无纺材料及其制备方法
CN112210888B (zh) * 2020-10-23 2021-06-25 中原工学院 利于组织再生的聚乳酸弹性无纺材料及其制备方法

Also Published As

Publication number Publication date
KR101540845B1 (ko) 2015-07-31
KR20130138153A (ko) 2013-12-18

Similar Documents

Publication Publication Date Title
WO2013183976A1 (fr) Patch destiné à la régénération tissulaire, comprenant un échafaudage tridimensionnel poreux fibreux
Badylak et al. The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium
US9725693B2 (en) Three-dimensional scaffolds for tissue engineering made by processing complex extracts of natural extracellular matrices
Stankus et al. Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix
JP7535070B2 (ja) 虚血性疾患または虚血性損傷を処置するための心臓線維芽細胞由来細胞外マトリックスおよびその注射可能製剤
CA2692005C (fr) Support collagenique modifie par greffage covalent de molecules d'adhesion : procedes et applications pour l'ingenierie de tissu contractile, la therapie cellulaire en thoracique et cardiovasculaire
US20190060517A1 (en) Scaffold for cardiac patch
Kenar et al. A 3D aligned microfibrous myocardial tissue construct cultured under transient perfusion
Guan et al. Dual-bionic regenerative microenvironment for peripheral nerve repair
WO2016209062A1 (fr) Bio-encre à deux constituants, biomatériau 3d la comprenant et son procédé de préparation
WO2014152906A2 (fr) Intestin issu de l'ingénierie tissulaire
Takanari et al. Skeletal muscle derived stem cells microintegrated into a biodegradable elastomer for reconstruction of the abdominal wall
Georgiadis et al. Cardiac tissue engineering: renewing the arsenal for the battle against heart disease
WO2020204230A1 (fr) Composite de nanofibre et d'hydrogel, et échafaudage pour régénération tissulaire le comprenant
Verdiyeva et al. Tendon reconstruction with tissue engineering approach—a review
Xu et al. Optimizing anisotropic polyurethane scaffolds to mechanically match with native myocardium
Li et al. Highly interconnected inverse opal extracellular matrix scaffolds enhance stem cell therapy in limb ischemia
Ge et al. Decellularized extracellular matrices for tissue engineering and regeneration
CN101500508A (zh) 生物分子连接的仿生支架
WO2018117569A1 (fr) Feuille de cellules souches cardiaques multicouche et son procédé de fabrication
WO2018117573A1 (fr) Feuille de cellules souches de crête neurale multicouche et son procédé de fabrication
KR20120063619A (ko) 섬유형 다공성 삼차원 지지체를 포함하여 구성되는 손상된 조직 재생용 패치
Chen et al. Using cell-seeded electrospun patch for myocardial injury: in-vitro and in rat model
US20210308335A1 (en) Fibrous 3-Dimensional Scaffold Via Electrospinning For Tissue Regeneration and Method For Preparing the Same
Roacho-Pérez et al. Chitosan Scaffolds for Cardiac Tissue Engineering

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13800263

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13800263

Country of ref document: EP

Kind code of ref document: A1