WO2012077891A1

WO2012077891A1 - Patch for regenerating damaged tissue and configured with a fibrous porous 3d support

Info

Publication number: WO2012077891A1
Application number: PCT/KR2011/006463
Authority: WO
Inventors: 이승진; 심인경; 양영일; 장양수; 정미라; 정혜진
Original assignee: 이화여자대학교 산학협력단
Priority date: 2010-12-08
Filing date: 2011-08-31
Publication date: 2012-06-14
Also published as: KR20120063619A; KR101226629B1

Abstract

The present invention relates to a patch for regenerating damaged tissue and configured with a fibrous porous 3D support of a biodegradable polymer material, which has cells or a drug (for regenerating damaged tissue) sheathed in a support and is capable of being attached directly onto the organ in which the damaged tissue is present. According to the present invention, the patch for regenerating damaged tissue is easy to handle and has high cell survivability after transplantation, so as to provide the treatment efficacy of a cell treating agent to a diseased region requiring cell transplantation. Accordingly, when the porous 3D support of a biodegradable polymer material, which has cells or a drug (for regenerating damaged tissue) sheathed in a support, is directly attached to an organ in which the damaged tissue exists, the success rate of cell transfer can be increased and the cells can move to ischemic areas to increase the capillary generation of the damaged tissue and thus accelerate tissue regeneration.

Description

【Specification】

[Name of invention]

Damaged tissue regeneration patch comprising fibrous porous three-dimensional support

The present invention is a damaged tissue regeneration comprising a fibrous porous three-dimensional support of a biodegradable polymer material that is seeded in the support cells or drugs for regeneration of the damaged tissue, which can be directly attached to the organ in which the damaged tissue is present. It is about a dragon patch. Background Art

In order to fully recover tissue and organ damage from an accident or disease, it is necessary to restore the function of the tissues and organs by using some black tissues of other bodies. However, there are many constraints on the application because it causes supply problems, immunity rejection, disease pollution, and social problems that are far short of demand.

In particular, the heart is known as a tissue that can not be recovered once damaged. Recently, cardiomyocyte division and slowing down of cardiomyocytes have been reported to occur in normal heart, but there is no way to increase the number of damaged cardiomyocytes. Moreover, in patients with severe late-stage heart failure, the only way to recover the heart is to use a heart transplant or ventricular assist device. Accordingly, cell transplantation therapeutics using cells have emerged as an alternative for the recovery of damaged tissues. Maximizing engraftment of cells after transplantation is critical for successful cell transplantation using cell transplantation therapeutics. It is known that cells with dividing ability do not progress to final differentiation than cells that have already been differentiated, and thus have good engraftment and red tropism after transplantation. In addition, the cardiomyocytes are characterized by consistent movement through electrical signaling with surrounding tissues.In order to transplant cells into the myocardium and induce improvement of cardiac function, the transplanted cells are well fused with the existing tissues to transmit the same signal. It is necessary to move into the system.

<7>

<8> Cells used for cell transplantation are myoblasts isolated from adult stem cells (myoblasts), cardiomyocytes, endothelial cells, fibroblasts, or stem cells that induce cardiovascular formation.

There are ongoing studies to replace dead heart tissue using skeletal myoblasts or muscle cells. This involves taking some of the muscle tissue from the patient, isolating and propagating the myoblasts and implanting them into the heart. These cells do not form intercellular gap junctions, which poses the risk of causing paravenous veins.

<10>

Recently, as the study of the enjoyment cells is activated, the transplantation of undifferentiated stem cells to the site of myocardial injury has been reported to improve the heart function. Clinical trials are underway to test the use of autologous bone marrow-derived cells for the treatment of myocardial infarction for the purpose of developing a cell population capable of cardiac regeneration.

(Per in et al., Circulation 107: 2294, 2003; Strauer et al., Circulation 106: 1913, 2002; Zeiher et al..Circulation 106: 3009, 2002; Tse et al., Lancet 361: 47, 2003; Stamm et al., Lancet 3661: 45, 2003). The test assumed that the cells could have a purifying function to improve blood perfusion of cardiac tissue.

In addition, clinical trials are under way to test the use of autologous skeletal muscle myoblasts for cardiac treatment (Menasche et al., J. Am. Col 1. Cardiol. 41: 1078, 2003; Pagan i et al. J. Am. Coll. Cardiol. 41: 879, 2003; Hagege et al., Lancet 36l: 491, 2003). However, it is not clear whether the contraction of striated muscle cells can function properly with the heartbeat.

<13>

<14> The method of administering the stem cells as described above is intracardiac injection of heart patients

(intramyocardial delivery), surgically injected directly into the myocardium, catheter injection into the coronary artery, and systemic administration such as venous vascular injection. However, the bioretention of the injected cells is very low, and it is reported 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 tissues, the incidence of infusion of cardiomyocytes must be increased to the patients, and the patients are in a situation of increasing pain. In addition, 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 if a high concentration of cells is transplanted, the cells remain in a poor environmental condition until they differentiate from the transplanted site. There is a limit to seeing the therapeutic effect of the wound.

<15>

When implanted in the heart tissue, it is considered to be implanted in the form of a cell sheet. The cell sheet refers to a single cell layer, and it is known that neonatal cardiomyocytes can be made into a single layer and can be stacked in layers up to three layers in vitro (Non-Patent Document 11: FASEB J 2006 Apr; 20 (6): 708-10). Recently, various cell sheets have been produced by using a temperature sensitive culture plate in which poly (N-isopropylacrylamide) (PI m) is applied to a commercially available polystyrene culture plate using an electron beam. In addition, the stacking of cell sheets has already been reported to develop a myocardial tissue mass that can be used as a graft (Japanese Patent Publication No. 2003-38170, International Publication No. 01/068799, Shimizu et. Fabrication of pulsatile cardiac tissue grafts using a novel 3D dimensional cell sheet manipulation technique and t emper at ur espons i ve cell culture surface: CircRes. 2002; 90: _e 40-e48). The myocardial tissue prepared in this way has been confirmed to have the same electrical activity as normal myocardial tissue in vitro and in vivo (in vivo).

By the way, the method of manufacturing the cell sheet using the temperature sensitive culture plate is relatively stable when the primary culture is performed in a strict manner so as to be optimal for the culture in the special culture plate. When the method conventionally practiced at the facility was applied as it was, sheeting of the cells was difficult. In addition, the cell sheet is pressed by using a method of laminating cell sheets, resulting in low cell viability, and a problem in that the cell sheet can not be made thicker without the angiogenesis due to the limitation of oxygen permeability. In addition, it is not yet possible to produce any cell sheet to fit the size of the affected tissue of the heart.

<18>

Therefore, in order to transplant a large amount of cells and improve oxygen permeability, it is preferable to use a method of transplanting cells using a support. The nature of the support is important to have a good function and a certain degree of mechanical strength in order to seed and culture the cells in order to deliver them to organs such as the heart. That is, it is necessary to stably retain and engraft cells in a uniform distribution state during culture, and to ensure good proliferation viability. In addition, it must be possible to fix the suture when transplanting to the affected area after culture. In addition, mechanical to withstand the compression of the heart beat It is necessary to use a support having strength.

<20>

Many cell transplantation techniques using scaffolds have been reported. In an attempt to mix cells with macromolecules and increase cell engraftment in the form of scaffolds, Leor et al. (Pharmacology & Therapeutics. 2005; 105: 151-163), and Cannizzaro et al. Introduced endogenous cell adhesion to polyethylene glycol copolymers by introducing RGD peptide sequences that induce cell adhesion to polyethylene glycol copolymers. Attempts have been made to help myocardial tissue regeneration by augmenting it (Biotechnol Bioeng. 1998; 58: 529-535).

In addition, Piao et al. Induced myocardial infarction (MI) in mice and cultured bone marrow-derived cells on glycolide and caprolactone-based copolymers that possess biocompatibility and then contained bone marrow-derived cells. It has been reported that implantation of high molecular weight polymer into the myocardial infarction area results in improved cardiac function (Biomaterials. 2007; 28: 641-649).

<23>

However, the study described the initial stages of the polymer polymer and the polymer support, and further study is needed. The scaffolds used in this study are single-layered membranes that do not have pores for open structures, and there are problems of cell viability using two-dimensional matrix scaffolds, If the method is used, there is a problem in handling such as tearing. In addition, in the case of the heart, since the cells of high density have to be transplanted, the pores of the support should be large and the porosity should be more than 90%, there was a problem in applying.

In particular, the support used by Piao et al. In the experiment was a sponge type support, which had a small porosity and difficult cell transplantation to the inside of the support. In addition, this has a problem that it is difficult to apply directly to a material having a curved surface of an organ such as the heart because of its constant shape.

<26>

Therefore, the present inventors use a fibrous support seeded with cells through three-dimensional culture instead of a method of transplanting a two-dimensional cell culture product, which is a conventional method, to generate a patch shape having a constant thickness, and to produce cells. After seeding, it is attached directly to the heart, which can be attached to organs without difficulty in handling, prevents invasion of inflammatory cells, and has properties close to the original tissues that are compatible with surrounding tissues. In addition, it was confirmed that the therapeutic effect of cell transplantation significantly improved and completed the present invention.

[Content of invention]

[Technical problem]

SUMMARY OF THE INVENTION An object of the present invention is to provide a patch for damaged tissue regeneration that increases the rate of cell delivery success and increases the angiogenesis of damaged tissue by promoting the cell transfer to the ischemic site and promotes tissue regeneration for the treatment of damaged tissue.

Technical Solution

In order to achieve the above object, the present invention provides a fibrous porous three-dimensional support of a biodegradable polymer material, which is seeded in the support for regeneration of damaged tissue or drug, and can be directly attached to the organ in which the damaged tissue is present. Provides a patch for damaged tissue regeneration, including

Advantageous Effects

Damaged tissue regeneration patch according to the present invention is easier to handle (handling) compared to the method of injection, single cell (single cee) and two-dimensional scaffold form, high cell survival residual after transplantation and transplantation Therapeutic efficacy of cellular therapies can be enhanced at the disease site as needed.

Therefore, if the fibrous porous three-dimensional scaffold of biodegradable high molecular material seeded with cells or drugs is directly attached to the organ where damaged tissue is present for the treatment of damaged tissue, the cell transfer success rate is increased and the cells move to the ischemic site. Increases angiogenesis of tissues and has the effect of promoting tissue regeneration.

[Brief Description of Drawings]

1 is an (a) photorealistic image and (b) differential scanning micrograph of the PLLA fibrous porous three-dimensional support patch prepared in Example 1;

FIG. 2 is a differential scanning micrograph of a PLLA fibrous porous three-dimensional support patch prepared with orientation;

Figure 3 is (a) a differential scanning micrograph attached to the heart stem cells attached to the randomly radiated non-oriented patch prepared according to Example 1, (b) having an orientation prepared according to Example 2 Is a differential scanning micrograph with cardiac stem cells attached to the patch;

4 is a cell transplanted into a fibrous porous three-dimensional support patch having an orientation, and after 48 hours H & E staining (a) a microscopic view of the cross-sectional cross section, (b) a cross-sectional view of the microscope;

5 is the number of cells measured on days 1, 4, 7, and 14 according to Experimental Example 2;

FIG. 6 is a H & E, aSA, HC, TnT immunostaining photograph of the stem seeded with cardiac stem cells according to Experiment 3 for 2 weeks in vitro in (a) growth medium composition and (b) differentiation medium composition;

7 (a) and 7 (b) are micrographs obtained by H & E staining of tissues after 4 days of implanting a patch seeded with cardiac stem cells on the outer pericardial surface of normal heart tissues according to Experimental Example 4;

Figure 8 is (a) a picture immediately after implanting the patch without seeding the stem cells of Comparative Example 1 in the myocardial infarction, (b) a tissue photograph after 14 days, (c) the heart of Example 1 in the myocardial infarction Pictures immediately after transplanting the seeded patch with stem cells and (d) tissue pictures after 14 days; 9 is a photograph obtained by H & E staining of the tissue obtained after 14 days according to Experimental Example 5.

(a) a transplanted picture of the patch of Example 1 seeded with cardiac stem cells, (b) a transplanted picture of the patch of Comparative Example 1 without heart stem cells;

10 is a tissue photograph obtained after 14 days according to Experimental Example 5 (a) immunostaining tissue photograph (b) DAPI staining nuclei, (c) (a) and (b) superimposed ;

11 is (a) TnT immunostained tissue photograph of the tissue obtained after 14 days according to Experimental Example 5, (b) DAPI stained nuclei, and (c) (a) and (b);

12 is a (a) SMA immunostained small artery / vein photograph, (b) DAPI stained nuclei, (c) (a) and (b) of the tissue obtained after 14 days according to Experimental Example 5 Photographs;

FIG. 13 shows (a) CD34 immunostained capillary images, (b) DAPI stained nuclei, and (c) (a) and (b) superimposed on tissues obtained after 14 days according to Experimental Example 5. FIG. Photo;

14 is a low-magnification photograph of the patch and heart tissue 14 days after transplantation according to Experimental Example 5 (a) Photograph showing the transplanted Dil-labeled heart stem cells in red fluorescence.

(b) a DAPI stained photo of the nucleus, (c) a superposition of (a) and (b);

Figure 15 is a high magnification micrograph of the myocardial tissue showing the location of cardiac stem cells transplanted in the myocardial layer of myocardial infarction rat, the upper part of the patch attached to the lower 7 2011/006463 Increasingly, the inside of the myocardium is shown, red fluorescence of (a) represents Dil-labeled heart stem cells, blue fluorescence of (b) represents nuclei labeled with DAPI, and (c) (a) ) And (b) are overlapping pictures;

<5i> FIG. 16 is a photograph obtained by H & E staining of a tissue obtained after 28 days according to Experimental Example 6.

(a) a photo of the implant of the patch of Example 1 seeded with heart stem cells, (b) a photo of the transplant of the patch of Comparative Example 1 without heart stem cells;

17 is a histological analysis of tissue obtained after 28 days in accordance with Experimental Example 6 when the patch of Example 1 is implanted and the patch of Comparative Example 1 without stem cells is transplanted.

(a) Fibrous area measurement result, (b) Heart thickness measurement result.

<53>

[Best form for implementation of the invention]

Hereinafter, the present invention will be described in detail.

<55>

The present invention comprises a fibrous porous three-dimensional scaffold of biodegradable polymer material which is seeded in a scaffold for regeneration of damaged tissue or a drug and can be directly attached to an organ in which the damaged tissue is present. Provide a patch for damaged tissue regeneration.

<57>

In the patch for damaged tissue regeneration of the present invention, it is preferable that the cells for regeneration of the damaged tissue seeded in the support are 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 heart tissue, it is preferable to use cardiac stem cells that are well differentiated into cardiac cells, have a cardiac regeneration effect, and are able to move into heart tissue. In addition, the present invention uses the myocardial stem cells of the mouse heart, but is not limited to these, similar to the stem cells collected from all kinds of mammalian species including humans, rats, mice, mice, etc. Is applied.

<60>

When the seed seeded patch is implanted into the damaged tissue, it can be seen that the stem cells are differentiated into cells of the tissue. In particular, cardiac stem cells can be used to differentiate using cardiac specific markers. These markers include MHC myosin heavy chain, cTnl (cardiac troponin 1), cTnT (cardiac troponin T), a -cardiac actin, a— actinin and MLC2 (myosin light chain), but are not limited to these. Phenotypes of these cells can be assessed by rhythmic contractions and the expression of markers associated with the heart.

<62>

In addition, in the patch for damaged tissue regeneration of the present invention, a drug may be applied in the patch for rapid recovery of the damaged tissue. The drug may use a gene or regenerative protein that improves the performance of genes and stem cells involved in tissue regeneration.

<64>

In particular, the regenerative protein is VEGF-A, VEGF-A, VEGF-

B, VEGF-C, VEGF-D, VEGF-E, Neuropilin, (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-beta, IGF-1, osteopontin, iotropin, activin, endovirin -1 It is preferably one or more selected from the group consisting of, but is not necessarily limited thereto. Vascular endothelial growth factors may act independently or in combination with each other. In combination, angiogenesis factors act synergistically and are more effective than the sum of the effects of separate individual factors.

As described above, the drug is seeded in the support to allow drug delivery, thereby maximizing tissue regeneration effect, and in particular, by releasing it slowly during regeneration period, the effect can be enhanced.

<67>

The damaged tissue regeneration patch of the present invention is capable of regenerating tissue by attaching directly to most organs in which damaged tissue is required for cell delivery, and may be any organ that can be attached in the form of a patch. In particular, the damaged tissue may be attached to the heart or liver where the damaged tissue is present to regenerate the damaged tissue.

For example, when a patch containing cardiac stem cells is attached to the heart, the cardiac stem cells are differentiated into cardiomyocytes, and the transplanted cells move to surrounding cardiac tissues such as the peripheral myocardial infarction area where the cardiomyocytes are damaged to repair damaged tissue. It is renewable.

<70>

In the damaged tissue regeneration patch of the present invention, the porous 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. It is particularly preferred to be made of hydrolyzable polymers of α and β-hydroxycarboxylic acids. 9 2011/006463 The biodegradable polymers are polylactic acid (PLA), polyel-lactic acid (PLLA), polyglycolic acid (PGA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L-lactide-co-glycolide); biodegradable aliphatic polyesters such as PLGA), poly (caprolactone), diol / diacid aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone) ), At least one synthetic polymer selected from the group consisting of poly (hydroxybutyrate) and poly (hydroxy valerate), polylactic acid (PLA) having a molecular weight of 100,000 to 350,000 kD It is more preferable to use, and it is most preferred to use polyl-lactic acid (PLLA), but is not necessarily limited thereto. In the damaged tissue regeneration patch of the present invention, the porous three-dimensional support can be produced by spinning in the form of fibers having a diameter between semimicro and nano using an electrospinning method. The spun form has a mesh form in which fibers are entangled and provides a three-dimensional support containing suitable voids. The size and distribution of the pores is a very important factor in determining cell growth, and the pores must be inter-connected so that the nutrient solution penetrates evenly into the scaffold and the cells grow well.

The diameter of the voids is preferably 50 urn or more and 300 or less, most preferably 100 m or more. It is a structure that helps the growth of transplanted cells to the size that can easily penetrate the cells, discharge of nutrients and waste, and can easily grow blood vessels after transplantation. In addition, in the damaged tissue regeneration patch of the present invention, it is preferable that the porous three-dimensional retardation retains the porosity of 90 to 99% and maintains the three-dimensional support to improve the growth of cells for tissue regeneration.

Such a damaged tissue regeneration patch can be easily attached to the organ with the damaged tissue by a surgical method, cardiac stem cells are differentiated into cardiomyocytes, angiogenesis in the ischemic area is increased and blood vessels are formed in the transplanted tissue. In addition, the transplanted cells are moved to the surrounding heart tissue, which is effective in tissue regeneration of the myocardial infarction region lacking cardiomyocytes. Damaged tissue regeneration patch of the present invention can be used by the production method as described below.

Biodegradable polymer alone or mixed to dissolve in organic solvent to prepare spinning solution 10 PT / KR2011 / 006463 (step 1); Spinning the polymer solution using an electrospinner and simultaneously evaporating the organic solvent to form a polymer fiber into a patch in the form of a network having a three-dimensional structure having a thickness of 50 to 1 cm or less (step 2); And seeding the drug or cells in the patch (step 3).

<82>

In the step 1, any organic solvent used to prepare a spinning solution as a polymer solution may be used as long as it is a volatile organic solvent having a low boiling point. Chloroform, dichloromethane, dimethylformamide, di It is preferable to use oxane, acetone, tetrahydrofuran, trifluoroacetic acid, 1,1,1,3,3,3, -nucleofluoroisopropylpropyl, and it is preferable to use dichloromethane.

<84>

In step 2, the spinning solution is manufactured using an electrospinner, and the spinning process using the electrospinner is as follows. A constant current flows from the voltage generator to form an electric field between the nozzle and the collector, which is then charged into the spinning reservoir, and the polymer solution is spun by the force of the electric field and the pressure of the pump. The condition of the electrospinner is a radiation distance of 10 20 cm, voltage 10 ~ 20 kv, release rate of 0.05 ~ 0.15 ml / min is preferably, but not limited to.

<86>

When the polymer solution is spun onto the collector when the solvent is volatilized, the solvent is volatilized, and the electrostatic repulsive force is applied, and the fibers do not adhere to each other. It can be produced, and the spun form can be produced to have a three-dimensional shape containing a suitable pore in the form of a mesh entangled fibers, the thickness is 50 ~: L cm or less.

In addition, when the temperature and humidity affecting the spinning, the viscosity of the solution, and the volatility of the solvent have optimal conditions, the fibers are separated and integrated into a separate three-dimensional support. It is possible. Particularly suitable are co-solvents of highly volatile dichloromethane and very low solubility acetone. Networked patches integrated in the collector can be removed and expanded in an appropriate physical way to increase the voids.

<89>

In addition, in the patch forming step of step 2, a patch in the form of a network having a three-dimensional structure may be manufactured to have an orientation. Because the heart muscle is directional and has to withstand pressure, it is replaced by a collector of a cylindrical drum instead of a collector of a normal stainless steel plate and the rotational speed is reduced.

If it is more than 1000 rpm, you can obtain a network-oriented patch in one direction.

<92>

In step 3, the cells usable as seeding the drug or cells in the patch of step 2 include stem cells capable of inducing tissue regeneration, and cells capable of differentiation. Genes involved in genes, genes that improve stem cell performance, and regenerative proteins.

In the process of preparing the patch, a drug-impregnated patch may be prepared by mixing a drug solution of an aqueous phase with an emulsion method in a polymer solution, and spinning a laminating the mixed solution. The drug dispersed in the biodegradable polymer solution in the emulsion step according to the present invention is emulsified in a therapeutically effective amount in a biodegradable high molecular solution using a surfactant known in the art, for example, Span 80, in water-in-oil emulsion type Evenly distributed.

The drug implants the seeded patch into the damaged tissue and releases the protein it contains when exposed to the aqueous environment. At the beginning of the release, it diffuses out of the patch and releases, and as the support itself begins to disintegrate, the release increases from the missing gap. Release rates and modalities can be varied depending on protein concentration, flow / water ratio, and site of application.

<96>

Thus, the patch of the present invention prepared by the electrospinning method is excellent in cell retention and viability, and also cultures cells or progenitor cells derived from tissues in artificial environment and / or in vivo using this support. By doing so, it is possible to prevent invasion of inflammatory cells during biotransplantation and to produce three-dimensional cell conjugates having properties close to the original tissues that are compatible with surrounding tissues.

<98>

In addition, in the damaged tissue regeneration patch of the present invention, the patch of the present invention can be set to a size necessary for appropriately setting the thickness and complementing the affected area. The patch thickness of the present invention is preferably 50 to 1 cm. When applied to the heart, it is desirable to have a thickness of 1 to 3 mm. There is no particular limitation on the shape and area of the support, and it is possible to manufacture a sized layer to cover the ischemic area. <ioo> Further, in the case of the conventional three-dimensional support is made in the form of a sponge bar adhesion strength is reduced due to the property of restoring to the original state when attached to the damaged tissue, the patch according to the present invention is a porous three-dimensional support angle fiber form bar damaged site Excellent adhesion to

<101>

[Form for implementation of invention]

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the present invention, the content of the present invention is not limited to the following examples.

<103>

Example 1: Preparation of a fibrous porous three-dimensional support patch containing stem cells 1

1) Preparation of a fibrous porous three-dimensional support patch

<106> The polyl-lactic acid (PLLA) was dissolved in a solvent of dichloromethane / acetone (acetone volume ratio 10¾-40% 부피 volume ratio 20% accurately tested) and the polymer concentration was 14-20% (exactly tested concentration 1). The solution was prepared to make.

<107> Using a DH High Voltage Generator (model name CPS-40K03VIT), an electrospinner manufactured by Chungpa EMT (Korea), the solution discharge rate was 0.06 ml / min, the voltage was 10 kv, and the distance of the electric field was 15 cm. It was electrospun into. The thickness of the fiber is about 7μη in diameter by electrospinning under conditions of 15 ~ 25 humidity 10 ~ 40% so that all solvent is volatilized before the fiber is accumulated on the stainless steel plate collector. ， A patch in the form of a fibrous network having a thickness of about 300 μηι was prepared, and then the pores were enlarged by physical expansion to prepare a patch using a fibrous porous three-dimensional support having a thickness of 1 mm (FIG. 1).

<108>

2) Seeding of Heart Stem Cells

In order to seed cardiac stem cells on the fabricated porous porous three-dimensional scaffold, 70% ethane was sterilized and washed with a buffer. After removing all the remaining water, ιχιο ⁶ ~ ιχιο ⁸ (5X10 ⁶ in this example) was attached to the support medium for more than one hour after inserting the suspended media to the support. Thereafter, incubation for 48 hours at 37 ^° C, 5% C02 conditions in the medium to stabilize the cell adhesion. Cells in Dulbecco's and 1% in the quality Eagle's medium (Dulbecco's Modified Eagle's Medium) penicillin and 10% fetal bovine serum, 10 ng / mi human EGF was added by using the prepared culture medium, respectively ⁵⁾ 37 ° under a carbon dioxide concentration Incubated at C saturated humidity. Example 2 Preparation of a Fibrous Porous Three-Dimensional Support Patch Containing Stem Cells 2 A patch in the form of a fibrous network oriented in one direction was prepared by replacing the collector with a cylindrical drum in a stainless steel plate and rotating at 2000 rpm. Except for producing a patch using a porous three-dimensional support containing stem cells in the same manner as in Example 1 (Fig. 2). Comparative Example 1 Patch with No Cells Transplanted

Using the same method as in 1) in Example 1, a patch using a fibrous porous three-dimensional support that did not seed cardiac stem cells was used. Experimental Example 1 ： Cardiac Stem Cell Attachment Experiment

In order to observe the cell adhesion of the patch using the fibrous porous three-dimensional support of Example 1 and Example 2 to stabilize the cell adhesion for 48 hours as follows.

Unattached cells are washed away and removed with 2.5% glutaraldehyde solution.

Fixed for 20 minutes. After fixation, the samples were dehydrated sequentially with 7, 80%, 90% and 100% ethanol for 10 minutes each and thoroughly vacuum dried. The sample was then coated with gold. The results of the observation with the differential scanning microscope are shown in FIG. 3. It can be seen that the cells adhered well to the prepared support, and for the support having the orientation of Example 2, the orientation of the cells along the fibers having the orientation was confirmed (FIG. 3 (b)). In addition, 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. After paraffin pome, slides of 4 iim thickness were prepared and stained with H & E (Hematoxylin and Eosin) and observed under a microscope (FIG. 4).

It can be seen that the cells are seeded at a high density in the photograph of the transverse section (FIG. 4 (a)), and in the photograph of the cross section, the cells are present at a high density as well as outside. b)). This is because the patch using the prepared fibrous porous three-dimensional support is easy to penetrate the inside of the stem cells because the size of the pores is large. <125>

Experimental Example 2 Observation of In Vitro Cell Proliferation

The stem cells prepared in Examples 1 and 2 were seeded using a fibrous porous three-dimensional scaffold seeded on a fixed date for 1 week, 4 days, 7 days, and 14 days while culturing for 2 weeks in a growth medium composition. Cell counts were measured by Cell Counting it-8 (CCK-8) analysis (FIG. 5). As shown in FIG. 5, both randomly radiated non-oriented patches (Example 1) and oriented patches (Example 2) were confirmed to have excellent cell proliferation ability.

<128>

Experimental Example 3 Observation of In Vitro Cell Differentiation

The patch seeded with the cardiac stem cells prepared in Example 1 was cultured in vitro for 2 weeks in the growth medium composition and cardiac cell differentiation medium composition. After fixing with paraffin to observe the differentiation into heart cells, the slides were made vertically and stained with H & E to obtain an image (FIG. 6).

<131> Cell orientation was observed in a specific direction and α-sar comer ic actinin (aSA), a myosin heavy chain, a sarcomere protein involved in cardiomyocyte contraction in the cell. (myosin heavy chain (MHC) and troponin-T (troponin- T, TnT) expression could be confirmed.

The degree of differentiation in the patch cultured in the differentiation medium was high, and the differentiation into cardiomyocytes was also observed in the culture medium in the growth medium. This may be a result of using stem cells present in the heart.

<133>

Experimental Example 4 Observation of Tissue Fusion and Inflammation by Normal Transplantation

<i35> Sprague-Daw ley rats (8-9 weeks) were anesthetized with isofurane. Anesthetized rats maintained breathing under ventilated under positive pressure. In order to observe the stability of the patch using a stem cell seeded porous true dimensional support, 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 tissues and the degree of inflammatory response after H & E staining are shown in FIG. 7.

In the region P (Periphery) of FIG. 7, the circled portion indicates blood vessels, and as a result, growth of blood vessels is formed in the patch. In other words, you can see that the fusion of the patch and the heart tissue is made well. In addition, with respect to cell differentiation, tissue formation at the interface between normal heart tissue and the patch (area I in FIG. 7) and at the periphery (area P in FIG. 7) As observed, fusion with cardiomyocytes can be observed.

<137>

Experimental Example 5 Analysis of Cardiac Function Improvement by Patch Transplantation Using a Porous Three-Dimensional Support Seeded with Heart Stem Cells in a Disease Model 1

1) Manufacture of myocardial infraction (MI model)

Spra ague-Daw ley 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.

<i4i> After performing ligation of left anterior Descending (LAD) and confirming ischemic site generation, the patch of Example 1 of 10x10 mm sized stem cells and the patch of Comparative Example 1 Cells without seeding were attached to the infarcted myocardium zone and the infarction border zone with 7-0 silk, respectively, of the epicardium surface. Immediately after the wound, the patches of Example 1 and Comparative Example 1 were implanted into the anterior wall of the left ventricle, regenerated 14 days after implantation, and the cardiac tissue was fixed with 10% buffered formaldehyde. Observation was made by paraffin sections.

(A) of FIG. 8 shows a photograph immediately after implanting the patch seeded with the cardiac stem cells of Example 1 in the myocardial infarction portion, and (b) shows a tissue photograph 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 portion and (d) shows the histological image after 14 days.

<143>

<144> 2) Analysis of cardiac function improvement results

(1) The tissue prepared in 1) was prepared as a slide and stained with H & E (hematoxylin and eosin) (FIG. 9). After 14 days of coronary artery ligation, myocardial infarction was successfully induced, and myocardial infarction and cardiac dilatation were observed.

As shown in FIG. 9, the patch of Example 1 and Comparative Example 1 was found to be completely fused with the LV anterior wall. In addition, when the implanted patch of the heart stem cells of Example 1 (Fig. 9 (a)) the shape of the transplanted patch is well 16 2011/006463 It can be seen that the LV anterior wall thickness of the heart is thicker than the heart of Comparative Example 1 implanted with a cellless patch. In addition, the heart implanted with the patch of Example 1 had less LV dilatation, and myocardial regeneration (white arrow) was prominent.

(2) After 14 days, the tissue transplanted with the cardiac stem cell seeded patch was stained with myocardial antibody and stained with a fluorescein isothiocyanate-conjugated secondary antibody. . These represent a-sarcomer ic actinin (aSA) and troponin-T (TnT) cardiomyocyte markers, which are sarcomere proteins involved in cardiomyocyte contraction.

10 (a) shows an aSA immunostained photograph and (b) shows all nuclei labeled with DAPI. 10 (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). In addition, FIG. 11 (a) shows a TnT immunostained photograph and (b) shows all nuclei labeled with DAPI. (C) of FIG. N shows that stem cells in the patch differentiated into TnT cardiomyocytes 14 days after the patch was implanted into the tissue as a result of overlapping (a) and (b).

(3) Smooth muscle alpha actin (SMA) and CD34 immunostaining showed that angiogenesis was increased in the hull region where the patch of Example 1 was transplanted 14 days after transplantation.

Figure 12 (a) is SMA immunostained small artery / vein picture and (b) shows all nuclei labeled with DAPI. 12 (c) shows that the small artery / vein grows in the patch as a result of overlapping (a) and (b).

In addition, FIG. 13 (a) shows CD34 immunostaining of capillaries and (b) shows all nuclei labeled with DAPI. FIG. 13C shows that capillaries grow and grow in the patch as a transplant tissue as a result of superimposing (a) and (b).

(4) To track the survival and progression of the cells after transplantation, cells were labeled with Dil, a red fluorescent marker at the time of cell collection, and approximately 10 ⁶ cells were transplanted into patches. 17 T / K 2011/006463 implanted in the infarct of the heart. Survival of stem cells after transplantation was determined by post fluorescence microscopy. 14 and 15 are a series of micrographs showing the location of transplanted heart stem cells in the myocardial layer of myocardial infarction rat 14 days after transplantation.

14 is a low magnification photograph of the patch and heart tissue 14 days after transplantation.

14 (a) shows red fluorescence of transplanted Dil-labeled heart stem cells.

(b) is a DAPI stained picture of the nucleus. (c) is a photograph in which (a) and (b) are superimposed and Dil positive cell aggregates are observed in the patch. This confirms that pleasant cells survive in the long-term patch and migrate into the myocardium.

15 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 of the patch attached and the lower down the inner myocardium Indicates. Red fluorescence in (a) represents Dil-labeled heart stem cells and blue fluorescence in (b) represents nuclei labeled with DAPI,

(c) is a superimposition of (a) and (b). Stem cells labeled with Dil were also found in the myocardial infarction area where the myocardial layer and the myocardial cells were missing. It confirmed that it moved.

<160>

Experimental Example 6 Analysis of Cardiac Function Improvement by Patch Transplantation Using a Porous Three-Dimensional Support Seeded with Heart Stem Cells in a Disease Model 2

1) Preparation of disease model (myocardial infraction, MI mode 1)

<i63> A disease model was prepared in the same manner as in Example 1, except that the patches of Example 1 and Comparative Example 1 were regenerated 28 days after implantation into the anterior wall of the left ventricle.

<164>

2) Analyzing the Cardiac Function Improvement Results

(1) The tissue prepared in 1) was prepared as a slide and stained with H & E (hematoxylin and eosin) (FIG. 16). After 28 days of coronary artery ligation, myocardial infarction was successfully induced, and myocardial infarction and cardiac dilatation were observed.

As shown in FIG. 16, when the patch seeded with the heart stem cells of Example 1 was implanted, the shape of the transplanted patch was well maintained, and the LV anterior wall thickness of the heart was a cellless patch. It can be confirmed that it is thicker than the heart of Comparative Example 1 implanted. In addition, the heart implanted with the patch of Example 1 was then left ventricular dilatation (LV) 18 2011/006463 dilatation was low, and regeneration (arrow area) of myocardium was prominently observed (FIG. 16).

<168>

(2) According to 1), the fibrosis area and the heart thickness of the tissues implanted with the patch of Example 1 and Comparative Example 1 were measured after 28 days. In the case of implanting the patch seeded with the cardiac stem cells of Example 1, it can be seen that the fibrotic area was significantly reduced (FIG. 17A). In addition, in the case of implanting the patch seeded with the heart stem cells of Example 1, LV anterior wall thickness can be confirmed to be thicker than the heart of Comparative Example 1 in which the cellless patch was implanted (FIG. 17). (B)).

Claims

[Range of request]

[Claim 1]

A patch for regenerating damaged tissue, comprising a fibrous porous three-dimensional support made of biodegradable polymer material, in which cells or drugs for regeneration of damaged tissue are seeded in a support, and directly attached to an organ in which the damaged tissue is present.

[Claim 2]

2. The patch for damaged tissue regeneration according to claim 1, wherein the cells are stem cells.

[Claim 3]

According to claim 1, wherein the drug is a damaged tissue regeneration patch, characterized in that the gene involved in tissue regeneration, a gene or a regenerative protein to improve the performance of stem cells.

[Claim 4]

The method according to claim 13, wherein the regenerative protein is (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-beta, IGF-1, Osteopontin, Piotropin, Activin, Endorphin-1 is one or more selected from the group consisting of patches for damaged tissue regeneration.

[Claim 5]

According to claim 1, wherein the organ is damaged tissue regeneration patch, characterized in that the organ is required for cell delivery for tissue regeneration and the patch can be attached.

[Claim 6]

The patch of claim 5, wherein the organ is a heart or liver.

[Claim 7] The method of claim 1, wherein the biodegradable polymer is polylactic acid (PLA), polyglycolic acid (PGA), poly-lactic acid (PLLA), poly (D, L-lactic acid-co-glycolic acid) (poly (D, L-lact ide-coglycolide; PLGA), poly (caprolactone), diol / diacid based aliphatic polyester, polyester-amide / polyester-urethane such as biodegradable aliphatic polyester and poly (ballet) Rolactone), poly (hydroxybutyrate) and poly (hydroxy valerate) is at least one synthetic polymer selected from the group consisting of patches for damaged tissue regeneration.

[Claim 8]

The patch for damaged tissue regeneration according to any one of claims 1 to 7, wherein the porous three-dimensional support has a pore diameter of 50 urn or more and 300 ίΜ or less.

[Claim 9]

The patch for damaged tissue regeneration of claim 1, wherein the porous three-dimensional support has a porosity of 90 to 99% to maintain growth of cells for tissue regeneration by maintaining the three-dimensional support.

[Claim 10]

The method of claim 1, wherein the fibrous porous three-dimensional support is a step of preparing a spinning solution by alone or mixing the biodegradable polymer dissolved in an organic solvent (step 1);

Spinning the polymer solution using an electrospinner and simultaneously evaporating the organic solvent to form a polymer fiber into a patch having a three-dimensional network shape having a thickness of 50 to 1 cm or more (step 2); And

Damaged tissue regeneration patch, characterized in that it is prepared by the step of seeding the drug or cells in the patch (step 3).

[Claim 11]

The patch for damaged tissue regeneration according to any one of claims 1 to 10, wherein the patch has a thickness of 50 im or more and 1 cm or less.