KR102001120B1 - Complex of nanofiber and hydrogel and a scaffold for tissue regeneration - Google Patents

Abstract

One aspect of the present invention relates to a nanofiber;
A first hydrogel layer encapsulating the nanofibers in a first hydrogel; And
A composite of nanofibers and hydrogels comprising a second hydrogel layer encapsulating the first hydrogel layer in a second hydrogel, a scaffold for tissue regeneration comprising the complex, and a method of manufacturing the same.

Description

TECHNICAL FIELD [0001] The present invention relates to nanofibers and hydrogels, and to scaffolds for regenerating tissues containing the nanofibers and hydrogels,

The present invention relates to nanofibers and hydrogel complexes and scaffolds for tissue regeneration comprising the nanofibers and hydrogels. More particularly, the present invention relates to nanofibers and hydrogel complexes that can be effectively used for regeneration of living tissue, A scaffold for tissue regeneration, and a manufacturing method thereof.

Cardiovascular diseases such as coronary artery disease, cardiac arrhythmia and heart failure have soared due to westernized eating habits and lack of exercise and aging, and deaths currently attributed to heart disease have the second highest mortality rate among major causes of death, Tissue regeneration is very important.

Since cardiovascular and myocardial tissue damage is difficult to treat and regeneration does not occur well, heart transplantation or cell transplantation therapy has been used for treatment. However, in the case of heart transplantation, the number of donors is significantly less than the number of patients required, and the cost of surgery is also a burden on patients. In addition, the cell transplantation treatment method is a method of allografting myocardial cells into damaged tissue. However, it is impossible to propagate as many myocardial cells as necessary until a sufficient amount of time is reached.

In order to solve the above problem, a method of regenerating tissue by making an artificial tissue out of the cell using the cell and effectively transplanting it into the body has recently been widely spotlighted, and studies have been actively conducted.

Patent Document 1 discloses a method for encapsulating human pancreatic cells and PDX1 (duodenal homeobox gene 1) positive human pancreatic progenitor cells using a polyethylene glycol hydrogel-based device, which is a biocompatible polymer. According to this, there is an advantage that the encapsulated precursor cells are well differentiated. However, since the differentiation is controlled by using the differentiation medium, physical properties such as biodegradability of the hydrogel can not be easily controlled.

Patent Document 2 discloses a method for encapsulating PDX1-positive human pancreatic progenitor cells by producing a three-dimensional cell encapsulation apparatus using PDMS (polydimethylsiloxane). This method is effective for cell differentiation, but due to the nature of PDMS, which is a constituent of the device, cell attachment ability is poor and it is difficult to regulate physical properties by modification.

In order to solve such problems, a method of encapsulating cells by preparing a complex of nanofibers and hydrogel by electrospinning has been developed. Patent Document 3 has devised a hydrogel composite of nanofibers and sodium alginate by electrospinning. The nanofibers were used as the cell adhesion layer, and the nanofibers were encapsulated with hydrogel to prepare a scaffold for tissue regeneration. However, due to the characteristics of the alginic acid hydrogel, there is a problem in that it can not propagate smoothly to cells that are adherent to proliferation, and thus it is difficult to generate blood vessels which are very important for tissue regeneration.

US Patent Registration 8278106 United States Patent Publication 20140257515 Korea patent registration 1685248

It is an object of the present invention to provide a complex that can be used as an effective tissue regeneration scaffold which can promote vascular formation important for tissue regeneration and have physical properties suitable for tissue transplantation.

Another object of the present invention is to provide a scaffold for tissue regeneration comprising the complex.

It is another object of the present invention to provide a method for producing the composite.

One aspect of the present invention is

Nanofiber;

A first hydrogel layer encapsulating the nanofibers in a first hydrogel; And

And a second hydrogel layer encapsulating the first hydrogel layer within the second hydrogel.

Another aspect of the present invention provides a scaffold for tissue regeneration comprising a composite of nanofibers and a hydrogel according to the above aspect.

In another aspect of the present invention,

Forming a first hydrogel layer for encapsulating the nanofibers by adding a nanofiber into a mold, adding a first hydrogel precursor solution, and then performing a crosslinking reaction; And

Forming a second hydrogel layer for encapsulating the first hydrogel layer by adding a second hydrogel precursor solution to the first hydrogel layer and then performing a crosslinking reaction to form a composite of the nanofibers and the hydrogel according to the first embodiment Of the present invention.

The complex of nanofiber and hydrogel according to one aspect of the present invention is characterized in that a tissue regeneration cell is cultured on the nanofiber by introducing a first hydrogel layer capable of adhering cells to the nanofiber, It is possible to cultivate vascular cells essential for tissue regeneration. Furthermore, when the complex is a scaffold for tissue regeneration and is transplanted into a living body for tissue regeneration, the fibrous bacterium as the scaffold and the fibroblasts of the living body as the scaffold and the second hydrogel layer, It is possible to block the fibrotic process by preventing the attachment of collagen and the like, thus enabling smooth transplantation. Therefore, the composite according to one aspect of the present invention can sufficiently avoid the rejection reaction of the living body such as fibrosis, which may occur in the scaffold transplantation process, by sufficiently forming blood vessels essential for tissue regeneration, For scaffolds, particularly scaffolds for cardiovascular tissue regeneration.

1 is a schematic view of a scaffold for tissue regeneration according to an embodiment of the present invention, which includes an electrospun nanofiber, a fibrin hydrogel layer encapsulating the nanofibers, and an alginate hydrogel layer encapsulating the fibrin hydrogel layer In the alginate hydrogel layer, biomolecules can be released and exchanged, proteins and cell invasion from the outside can be prevented or controlled, coexistence of cells can be achieved on the electrospun nanofibers, and fibrin hydrogel Vascular tissue formation is possible.
FIG. 2 is a schematic view of an embodiment of the present invention, including a process for preparing a nanofiber scaffold by electrospinning (step 1), disinfection of the scaffold (step 2), fibrin hydrogel encapsulation (step 3), and alginate hydrogel encapsulation (step 4) Fig. 2 is a schematic view showing a manufacturing process of a composite according to one embodiment of the present invention.
Fig. 3 shows the production process (a, b) of the nanofibers by electrospinning and the shapes (c, d) of the electrospun nanofiber scaffold through the scanning electron microscope.
Fig. 4 is a graph showing the results of a comparison between the fibrin hydrogel (a) prepared in Examples 1 to 3, the composite (b) of electrospun nanofiber and fibrin hydrogel, the electrospun nanofiber, the fibrin hydrogel, and the complex of alginate hydrogel c) taken with the naked eye.
FIG. 5 is a graph showing the transmittance of the electrospun nanofibers (Fibrin), the electrospun nanofibers and the complex of fibrin hydrogel (Fibrin-PCL), and the electrospun nanofiber, fibrin hydrogel, and alginate hydrogel prepared in Examples 1, Gel composite (Fibrin-PCL-alginate) of the present invention.
6 is a photograph of a cross section of the nanocage prepared in Example 3 using a cryo-scanning electron microscopy (Cryo-SEM).
FIG. 7 shows that HUVEC was dispersed in fibrin hydrogel at a concentration of 1 × 10 ⁵ cells / ml in a fibrin hydrogel without electrospun nanofibers for one week in order to confirm the viability of the HUVEC in the fibrin hydrogel. Survival was assessed by Live / Dead cell viability assay.
FIG. 8 is a graph showing the results of a comparison between a primary hydrogel and a secondary hydrogel in the presence of an HNFEC-containing fibrin hydrogel coated with an alginate hydrogel without electrospun nanofibers, followed by 1, 4, And 7 days after culturing, and then photographing a section of the two hydrogels through an optical microscope
FIG. 9 is a graph showing the results of FVH-containing fibrin hydrogel without radiofrequency nanofiber to confirm the suitability of fibrin hydrogel for angiogenesis. The fibrin hydrogel containing HUVEC was prepared at 1 day, 4 days, This is a photograph of the result of observing the process.
10 is a graph showing the results of measurement of cell activity by culturing HUVEC and ADSC on fibrin hydrogel and electrospun nanofibers for one week in order to observe cell activity in the nanocage according to one embodiment of the present invention to be.

Hereinafter, the present invention will be described in more detail.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. In addition, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. Also, the numerical values set forth herein are considered to include the meaning of " about " unless explicitly stated. Whenever a component is referred to as "comprising ", it is to be understood that the component may include other components as well, without departing from the scope of the present invention. The contents of all publications referred to in this specification are incorporated herein by reference in their entirety.

The present inventors have intensively studied scaffolds which are advantageous for culturing vascular cells capable of forming vascular tissues essential for tissue regeneration as well as culturing of tissue regeneration cells. As a result, they have found that nanofibers, A complex of nanofibers and hydrogels incorporating a hydrogel layer and a second hydrogel layer which encapsulates it again.

The present invention, in one aspect,

Nanofiber;

A first hydrogel layer encapsulating the nanofibers in a first hydrogel; And

And a second hydrogel layer encapsulating the first hydrogel layer within the second hydrogel.

In this specification, the composite of nanofiber and hydrogel according to one aspect of the present invention is simply referred to as "nanocage ".

Among the above complexes, the nanofibers are positioned at the innermost part of the complex. When the complex is used as a tissue regeneration scaffold, the nanofibers are used for culturing tissue regeneration cells, for example, co-culture of stem cells and myocardial cells Region.

The nanofiber is not particularly limited as long as the nanofiber is capable of culturing the tissue regeneration cell. In one embodiment, the nanofiber is electrospun nanofiber prepared by electrospinning. The electrospun nanofibers may be any electrospun nanofiber known to be suitable for the cultivation of conventional living cells.

The electrospun nanofiber is an aggregate of fibers having a diameter of several tens of nanometers to several micrometers. The structure includes a plurality of pores of various sizes, and forms a three-dimensional structure capable of cell attachment . These pores also have a predetermined shape, size and volume. The diameter of the electrospun nanofibers can be varied by adjusting the concentration of the polymer solution, the flow rate of electrospinning, the voltage, etc., which are the materials of the electrospun nanofibers, and may be changed by adding a salt to the polymer solution.

In one embodiment, the size of the pores of the electrospun nanofiber can be adjusted to 20 to 100 탆. Since most of the cells are fixed to the scaffold and are present at a size of 10 to 15 占 퐉 before transformation, tissue regeneration cells enter the pore in the above-mentioned size range and are fixed in the scaffold to be suitable for growth and differentiation have.

The electrospun nanofiber may be a nanofiber prepared by a known electrospinning method. The material used to produce nanofibers can be any material that is suitable for culturing tissue regenerating cells and capable of producing nanofibers. The scaffolds formed of nanofibers can undergo further modifications to ensure that the biomaterial is well immobilized on the nanofibers. For example, an oxygen plasma treatment, a radiation grafting method, or a selfassembled monolayer (SAM) method may be used for the additional modification. In the case of oxygen plasma treatment, nanofibers with high hydrophobicity are used to increase hydrophilicity. For example, nanoparticle scaffolds may be irradiated with UV radiation to produce benzophenone and azide compounds to modify the surface of the nanofibers to a desired material by a radiation grafting method. The SAM method uses a spontaneous reaction of a silane with a surface on which a hydroxy functional group exists to fix a portion bonded with the silane to the surface. Radiation grafting and SAM methods generally involve the use of N-hydroxysuccinimide (NHS), which is a chemical compound that binds to proteins. Additional modifications to the scaffolds formed with these nanofibers allow the biomaterial to be immobilized regardless of the type of nanofiber product, so there is no limit to the type of nanofiber product.

The type of the polymer forming the electrospun nanofibers is not particularly limited as long as it does not inhibit the culturing of tissue regeneration cells, and various natural polymers and synthetic polymers can be used. Polymer materials having no biodegradation characteristics in terms of cell attachment may be used, but polymer materials having biodegradation characteristics in terms of drug delivery, cell transplantation and cell treatment may be used. Such biodegradable polymers include, for example, chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), polylactic acid Poly (3-hydroxybutyrate) -co- (3-hydroxyvalerate) (PHBV), polydioxanone (PDO), poly ), Poly [(L-lactide) -co- (caprolactone)], poly (ester urethane) (PEUU) (PVA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polystyrene (PS), polyaniline (PAN) ≪ / RTI >

In one embodiment, the biodegradable polymer used in the preparation of the electrospun nanofiber is electrospun nanofiber of a mixture of PCL and gelatin. The electrospun nanofibers may also be suitable for adhesion, proliferation and differentiation of stem cells as well as tissue regeneration cells.

The nanofibers may be coated with Fibronectin for better adhesion of cells as needed. In addition, a growth factor used for facilitating cell proliferation and differentiation in terms of drug delivery can be introduced into the electrospun nanofiber. For this purpose, it may be mixed with the polymer solution during the electrospinning process and injected into the fiber, or physically / chemically attached to the outside of the fiber.

The electrospun nanofibers are prepared by electrospinning a solution of a polymer, which can be produced by a method commonly used in the art.

A polymer solution for electrospinning can be prepared by selecting a suitable solvent capable of dissolving the polymer as described above for the production of electrospun nanofibers. Such a solvent is not particularly limited as long as it satisfies the above conditions. For example, the solvent may be chloroform, 2,2,2-trifluoroethanol, TFE, Tetrahydrofuran (THF), dimethylform Dimethylformaldehyde (DMF), and any combination thereof, and can be suitably selected by those skilled in the art in view of factors such as the viscosity and dielectric constant of the solution.

In the electrospinning method for preparing the electrospun nanofibers, when a high voltage is applied to the polymer solution, one charge of + or - is accumulated in the polymer solution, and the mutual repulsion between the same charges exceeds the surface tension of the polymer solution As the solvent evaporates, the fibers are collected into a charged or grounded substrate with the opposite charge transferred to the solution. The diameter of the fiber can be controlled by adjusting the concentration, the flow rate, and the voltage of the solution in which the polymer is melted using the solvent, and the density of the fiber can be controlled by controlling the electrospinning time and the height of the nozzle and the substrate.

The solvent used for the polymer solution for the preparation of the electrospun nanofibers may have toxicity which is not suitable for the cultivation of tissue regeneration cells and is not completely evaporated during the electrospinning process, There is a concern. Therefore, in order to use the complex as a tissue engineering scaffold, it is necessary to completely remove the residual solvent. In order to remove the residual solvent, there is a method of storing the fibers at a temperature of several tens of degrees Celsius for evaporation of the solvent or storing the fibers in a vacuum state, and the above methods may be used in combination for more effective treatment. .

The nanofibers are encapsulated in a first hydrogel and then encapsulated in a second hydrogel to form the nanofiber and hydrogel complex.

The first hydrogel encapsulating the nanofibers may be formed by inserting the nanofibers into the first hydrogel to encapsulate the nanofibers. For the encapsulation, the nanofibers may be encapsulated in a first hydrogel by, for example, placing the nanofibers in a hydrogel precursor solution and crosslinking the hydrogel precursor solution to form a hydrogel. In addition, the nanofibers encapsulated in the first hydrogel may be encapsulated in a second hydrogel in the same manner.

The first hydrogel is a hydrogel suitable for culturing vascular cells required for angiogenesis because of its high cell adhesion ability. When the second hydrogel is transferred to a living body as a scaffold because its cell adhesion ability is poor, It is a hydrogel that can prevent external invasion of cells. That is, the first hydrogel has an excellent ability to adhere vascular endothelial cells and can greatly contribute to proliferation and angiogenesis, and the second hydrogel has a significantly smaller pore size than the first hydrogel, Although it can encapsulate cells, it does not help the proliferation of adherent proliferating cells, but it has the advantage of preventing the proliferation of non-adherent proliferating cells and the external invasion of proteins and cells during transplantation into the body.

Specifically, the first hydrogel may be a porous hydrogel having a pore size of 1 μm to 100 μm, and the second hydrogel may be a porous hydrogel having a pore size of 1 nm to 10 nm.

Since the hydrogel has a three-dimensional structure similar to that of the electrospun nanofiber, it helps to cultivate the cells in three dimensions. However, the hydrogel may exhibit a large difference in the cell adhesion ability depending on the characteristics of the polymer constituting the hydrogel. For example, polymers such as collagen exhibit good properties for adhesion of cells in the form of hydrogel. However, polymers such as PEG and alginate have a great restriction on cell adhesion when they are made from hydrogel, Suitable. Accordingly, the first hydrogel and the second hydrogel can be prepared by appropriately selecting the polymer. In addition, depending on the conditions for producing the hydrogel, the ability of the cells to adhere can be changed. Therefore, the cell adhesion ability of the hydrogel can be controlled by adjusting the porosity according to the manufacturing conditions, and a method known in the art Accordingly, it can be suitably manufactured by a person skilled in the art.

The first hydrogel may be formed of a biocompatible polymer suitable for culturing vascular cells required for angiogenesis, and may be selected from the group consisting of collagen, chitosan, fibrin, a copolymer thereof, and any combination thereof. Hydrogel of a biocompatible polymer, but is not limited thereto.

In one embodiment, the first hydrogel is a fibrin hydrogel.

Since the composite of the nanofibers and the hydrogel includes a porous first hydrogel layer, it is possible to adhere and cultivate the vascular cells, thereby making it possible to form blood vessels essential for tissue regeneration.

The second hydrogel may be formed of a biocompatible polymer capable of blocking cell proliferation. Examples of the second hydrogel include polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA) (PVA), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PAA), polyvinyl alcohol But are not limited to, lactones (PCL), gelatin, alginic acid, carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylate, polyvinyl chloride, maleic anhydride / And hydrogels of biocompatible polymers selected from the group consisting of any combination thereof, but are not limited thereto.

In one embodiment, the second hydrogel is alginic acid hydrogel.

Since the complex of the nanofibers and the hydrogel further includes a secondary hydrogel layer, when the complex is transplanted as a scaffold, it is possible to block the rejection reaction such as fibrosis. When transplanting into a living body using only scaffolds or nanofibers composed of only nanofibers and a scaffold made of porous first hydrogels, fibrosis occurs and the effect of transplantation of the extracorporeal tissue is very poor. As the second hydrogel, When the hydrogel is encapsulated and passivated, an excellent effect in transplantation in vivo can be obtained.

The first hydrogel layer and the second hydrogel layer may be formed by applying a hydrogel precursor solution and performing a crosslinking reaction. The polymers forming the hydrogel are variously crosslinked depending on their characteristics. For example, photo-crosslinking by ultraviolet (UV) irradiation, crosslinking using a reactive crosslinker, and the like can be cited. The crosslinked hydrogel using such a method is not easily decomposed, In the process, the mask can be used to cross-link the hydrogel to only a specific part, thereby grafting the patterning technique. In addition, a thermal crosslinking method in which crosslinking is carried out at a temperature is crosslinked at a specific temperature or higher to form a hydrogel, which can be applied to cell release and drug delivery through temperature change. In addition, the cross-linking method using the specific protein reaction and the reaction between the ion and the polymer has a merit that the hydrogel can be crosslinked using the reaction between the well-known proteins and the hydrogel can be easily manufactured.

In one embodiment, when the fibrin hydrogel is included as the first hydrogel, the fibrin hydrogel may be formed by using the fibrinogen as a hydrogel precursor solution and adding thrombin as a cross-linking agent. In addition, when the alginate hydrogel is included as the second hydrogel, the alginate salt solution can be used as a precursor solution such as calcium chloride to form the alginate hydrogel.

Since the hydrogel has a three-dimensional porous structure like the electrospun nanofibers, it can function as a drug delivery system through the release of drug from the inside. The hydrogel may be capable of sustained release of the drug. In order to introduce the drug into the hydrogel, it is possible to introduce the drug into the hydrogel after the addition of the drug before the cross-linking reaction for the hydrogel gel, and to continuously transfer the drug from the hydrogel. As another method for introducing the drug into the hydrogel, a drug may be added from the medium to the inside of the hydrogel by adding the drug to the external medium during the cell culture using the hydrogel.

In one embodiment, VEGF (Vascular Endothelial Growth Factor) or S1P (Sphingosin-1-phosphate), which is a growth factor that contributes to angiogenesis, may be mixed in the medium and transferred to the inside of the hydrogel, It can promote angiogenesis in the gel layer.

The composite of the nanofibers and the hydrogel may have sufficient strength not to be easily broken during transplantation in vivo for tissue regeneration. Since the composite includes a first hydrogel layer and a second hydrogel layer that encapsulate nanofibers, the strength of the composite may be improved depending on the crosslinking degree of the hydrogel, the thickness of the nanofiber, the thickness of the hydrogel, etc. Conventional descriptors can be adjusted according to known methods. Accordingly, when the complex has a sufficient strength, a scaffold for tissue regeneration similar to the physical strength of the extracellular matrix of each tissue to be regenerated can be produced. If the physical strength is similar, the tissue of the cells in the scaffold There is an advantage that specific specificity can be easily given,

In one embodiment, the composite may have an intensity of 0.1 to 0.5 MPa. If it is higher than the above strength, blood vessel regeneration may not be smoothly performed, and if it is lower, it may become difficult to control the differentiation of stem cells.

The complex may include tissue regenerating cells adhered to the nanofibers for use as a tissue regeneration scaffold.

The tissue regeneration cell may be any cells required for tissue regeneration, including cardiomyocytes, smooth muscle cells, cartilage cells, bone cells, skin cells, fibroblasts, vascular endothelial cells, neurons, Schwann cells , Stem cells, and any combination thereof. In one embodiment, cardiomyocytes and stem cells can be co-cultured on the nanofibers.

The first hydrogel layer may contain cells, biomaterials, or any combination thereof. The cell and / or the biomaterial may be any cell or biological material that can contribute to tissue regeneration. Such cells may be used in combination with interstitial cells selected from fibroblasts, vascular endothelial cells, neurons, Schwann cells, stem cells, and any combination thereof. The biomaterial may be a biomaterial capable of contributing to tissue regeneration, and may be, for example, a growth factor such as VEGF or S1P mentioned above which promotes the proliferation of vascular cells.

In one embodiment, the first hydrogel layer may contain human umbilical vein endothelial cells (HUVEC), which can promote angiogenesis by containing HUVEC, which can significantly improve tissue regeneration. Additionally, the first hydrogel layer may contain a growth factor, which may promote cell proliferation on the nanofibers due to the presence of growth factors and proliferation of vascular cells in the first hydrogel layer. In one embodiment, the growth factor is vascular endothelial growth factor (VEGF).

In one embodiment, the complex is a complex containing cardiomyocytes and stem cells adhered to nanofibers, wherein the first hydrogel layer contains HUVEC.

In one embodiment, the complex is a complex comprising cardiomyocytes and stem cells adhered to nanofibers, wherein the first hydrogel layer contains HUVEC and VEGF.

In another aspect, the present invention provides a scaffold for tissue regeneration comprising a composite of nanofibers and a hydrogel according to the above aspect.

The details of the tissue regeneration scaffold may be applied to the composite of the nanofiber and the hydrogel according to one aspect of the present invention.

As used herein, the term "scaffold" is an essential element in the field of tissue regeneration engineering, and refers to a structure that serves to provide an environment suitable for cell adhesion, differentiation, and proliferation and differentiation of cells migrated from around the tissue.

In the tissue regeneration scaffold, a regenerable tissue can be determined depending on the kind of cells adhered to the nanofibers. For example, cardiomyocytes for cardiovascular tissue regeneration, cartilage cells for cartilage tissue regeneration, bone cells for regeneration of bone tissue, skin cells for regeneration of skin tissue, and fibroblasts for wound tissue regeneration can be used.

In one embodiment, the tissue regeneration scaffold is a scaffold for cardiovascular tissue regeneration, and the nanofiber contains adherent cultured myocardial cells or, additionally, stem cells.

In one embodiment, the tissue regeneration scaffold is a scaffold for wound healing, and the nanofibers contain fibroblasts, or additionally stem cells.

Conventionally, when a scaffold containing cells is transplanted into a living body, many cases of transplantation failure due to fibrosis in vivo have been reported. However, the tissue regeneration scaffold according to the present invention can prevent the fibroblasts and collagen from adhering to the scaffold by the passivation due to the introduction of the secondary hydrogel layer, thereby blocking the fibrosis process, thereby allowing smooth transplantation .

In addition, the tissue regeneration scaffold can form vascular tissue by cultivating vascular cells in the first hydrogel layer, and can regenerate living tissue stably and effectively when the scaffold is transplanted in vivo.

Therefore, the complex according to one aspect of the present invention can sufficiently prevent the rejection reaction of the living body, such as fibrosis, which may occur in the scaffold transplantation process, by sufficiently forming blood vessels essential for tissue regeneration, Can be used as a scaffold, particularly as a scaffold for cardiovascular tissue regeneration.

In addition, the tissue regeneration scaffold is capable of introducing a desired drug into the electrospun nanofibers and hydrogel to enable continuous drug release, and by incorporating a drug effective for constructing an extracellular matrix (ECM) Can facilitate the formation of smooth extracellular matrix in the surrounding tissue. Due to the inclusion of these drugs, more effective applications are possible in areas such as wound healing.

1 is a schematic diagram of a tissue regeneration scaffold according to one embodiment of the present invention.

The tissue regeneration scaffold according to Fig. 1 has an electrospun nanofiber, a fibrin hydrogel layer encapsulating the nanofibers, and an alginate hydrogel layer encapsulating the fibrin hydrogel layer. In the alginate hydrogel layer, release and exchange of biomaterials can be performed, protein and cell invasion from the outside can be prevented or controlled, coexistence of cells can be achieved on the electrospun nanofibers, It is possible to create an organization.

According to another aspect of the present invention,

Forming a first hydrogel layer for encapsulating the nanofibers by adding a nanofiber into a mold, adding a first hydrogel precursor solution, and then performing a crosslinking reaction; And

Forming a second hydrogel layer for encapsulating the first hydrogel layer by adding a second hydrogel precursor solution to the first hydrogel layer and then performing a crosslinking reaction; Gel, comprising the steps of:

The method of preparing the composite of nanofibers and hydrogel according to an aspect of the present invention can be applied as it is to the composite of nanofiber and hydrogel.

In one embodiment, the first hydrogel precursor is fibrinogen and the cross-linking reaction can be cross-linked using thrombin as a cross-linking agent. Also, the second hydrogel precursor is an alginate aqueous solution, and the crosslinking reaction can be carried out using calcium chloride as a crosslinking agent. The alginate aqueous solution is, for example, sodium diacetate.

The nanofibers can be used after seeding a cell dispersion of tissue regeneration cells as described above on the nanofibers, followed by culturing.

In one embodiment, the cell dispersion is a mixed dispersion of cardiomyocytes and stem cells, the first hydrogel precursor solution may contain HUVEC, and may further contain VEGF.

FIG. 2 is a schematic view showing a step-by-step process of the method for producing the composite of the nanofibers and the hydrogel according to one embodiment. According to Fig. 2, the preparation of nanofibers by electrospinning (step 1), disinfection of nanofibres (step 2), formation of a fibrin hydrogel layer encapsulating nanofibers (step 3), and encapsulating the fibrin hydrogel layer And formation of an alginate hydrogel layer (step 4).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Formation of PCL-gelatin fiber layer by electrospinning

After mixing 0.5 g of polycaprolactone (PCL, Sigma) and 0.5 g of gelatin (Sigma) in 5 ml of trifluoroethanol (TFE, Sigma), the solution was completely sealed so that the solvent did not evaporate, I completely dissolved the gelatin. The polymer solution thus prepared was cooled at room temperature and then subjected to voltexing so as to be thoroughly mixed.

The solution was placed in a syringe and placed in an electrospinning system. The solution was then pumped at a constant flow rate of 0.7 ml / hr using a syringe pump to form a cylindrical needle tip. A voltage of 8 kV was applied to a cylindrical needle made of metal using a high-voltage power supply. A collecting plate for receiving the fibers obtained by electrospinning was grounded, and an aluminum foil was laid on the collecting plate. Then, a cover glass (size 1.8 cm x 1.8 cm) Cover glass was wrapped with aluminum foil on a dust collecting plate to obtain a fibrous sheet by electrospinning for 30 minutes. The distance between the cylindrical needle on which the voltage is applied and the dust collecting plate was about 10 cm. As the solvent of the polymer solution evaporated due to the voltage difference, the solution was pulled to the grounded dust collecting plate to form the fiber layer. The resulting electrospun nanofibers were left in a vacuum at about 60 DEG C for about 24 hours to remove residual solvent.

Figs. 3 (a) and 3 (b) are photographs of the electrospun fiber obtained by removing the residual solvent in a vacuum state using a cellphone camera, c and d show the results of coating the electrospun fiber with 15 mA of platinum for 1 minute, It is a photograph taken through a microscope.

Example 2: Fabrication of a composite of electrospun nanofiber and fibrin hydrogel

The electrospun nanofibers prepared in Example 1 were washed twice with phosphate-buffered saline (PBS) to completely remove residual solvent, and then cut into a size of 5 mm × 5 mm and placed in a 96-well plate.

To prepare a fibrin hydrogel precursor solution (ie, a fibrinogen solution), 0.1 mg of sodium chloride (NaCl) was dissolved in 5 ml of distilled water by vortexing at room temperature for 30 minutes. Fibrinogen human plasma, Sigma) was added and dissolved at room temperature for 10 minutes.

Thrombin was used as a crosslinking agent for crosslinking fibrinogen to fibrin. To prepare thrombin solution, 0.2 mmol of calcium chloride (CaCl ₂ ) was added to 5 ml of distilled water, and the mixture was vortexed at room temperature for 30 minutes. 100 U (Enzyme unit) was added to the solution and thrombin was dissolved at room temperature for 10 minutes to prepare a fibrin solution.

First, the electrospun nanofibers were placed in a 96-well plate, and the prepared fibrinogen solution was added to such an extent that the electrospun nanofibers were locked, and the prepared thrombin solution was added to the fibrinogen solution in an amount having a volume ratio of 1: 1, followed by pipetting Pipetting). The solution was stored at 37 ° C for 30 minutes for crosslinking of fibrinogen, thereby producing a composite of electrospun nanofiber and fibrin hydrogel.

Example  3: electrospun nanofiber, fibrin Hydrogel , And Alginate Hydrogel  Composite production

To prepare the alginate hydrogel precursor solution, 0.1 g of sodium alginate (Wako) was added to 5 ml of distilled water. The distilled water was sealed to prevent evaporation and completely dissolved at 37 ° C for 2 hours. Further, 0.1 g of calcium chloride (CaCl ₂ ) as a crosslinking agent was added to 10 ml of distilled water and completely dissolved at room temperature for 30 minutes.

100 μl of the alginate hydrogel precursor solution prepared using the above method was placed in a 96-well plate, and the complex of the electrospun nanofiber and fibrin hydrogel prepared in Example 2 was placed therein. Then, to form an alginate hydrogel, 50 μl of a calcium chloride solution as a cross-linking agent was placed in a sodium alginate aqueous solution and left at room temperature for 5 minutes. As a result, a nanocage, which is a composite of electrospun nanofiber, fibrin hydrogel, and alginate hydrogel, was prepared.

(C) of fibrin hydrogel (a), electrospun nanofiber and fibrin hydrogel complex (b), electrospun nanofiber, fibrin hydrogel, and alginate hydrogel prepared in Examples 1, 2 and 3, Is shown in Fig.

Experimental Example  1: fibrin Hydrogel , Electrospun nanofibers and fibrin Hydrogel  Composite and Nanocage Properties Evaluation

The fibrin hydrogel, the complex of the electrospun fiber and the fibrin hydrogel, and the nanocage made in the above conditions were washed with PBS solution to remove the residual hydrogel precursor, and then subjected to a compressive stress and a compressive strain It was placed on an instron (Instron corporation) equipment substrate for measurement.

Compressive stress and compressive strain were measured by compressing each scaffold to 80% of the existing height of each scaffold in the measurement of compressive stress and compressive strain. Compressive modulus was calculated by measuring the actual compressive stress and the actual compressive strain using the equation, and then measuring the slope of the graph in a certain section of the graph. The formula for calculating the actual compressive stress and the actual compressive strain is as follows.

s: measured compressive stress, e: measured compressive strain, σ: actual compressive stress, ε: actual compressive strain

FIG. 5 is a graph showing the results of a comparison between the prepared electrospun nanofibers (Fibrin), electrospun nanofibers and fibrin-hydrogel complexes (Fibrin-PCL) prepared in Examples 1, 2 and 3 and electrospun nanofibers, fibrin hydrogel, (Fibrin-PCL-alginate) of alginate hydrogel.

Experimental Example 2: Nano cage cross-section identification test

The section of the nanocage was observed. First, the nanocage was quenched by liquid nitrogen, and then cut vertically. The vertical cross section of the nanocage was observed using a cryo-scanning electron microscope (Cryo-SEM). The results are shown in Fig.

6 is a photograph of a cross section of the nanocage prepared in Example 3 using a cryo-scanning electron microscopy (Cryo-SEM).

6, cross-sections (a, b) between the fibrin hydrogel and the alginate hydrogel of the nanocage and cross-sections (c, d) between the fibrin hydrogel and the electrospun nanofiber were confirmed, and fibrin hydrogel Showed porosity, and the alginate hydrogel showed a flat cross-section.

Example 4: Fabrication of nanocage containing cells

The culture medium for culturing adipose-derived mesenchymal stem cells (ADSC) and cardiomyocytes was prepared by adding 10% FBS (Fetal bovine serum) and 1% penicillin (DMEM) to Dulbecco's Modified Eagle's Medium / streptomycin were used. The medium used for culturing human umbilical vein endothelial cells (HUVEC) was a mixture of 2% FBS and 1% penicillin / streptomycin in EGM-2 Bullekit (Endothelial Cell Growth Medium).

For co-culture of ADSCs and myocardial cells, a cell suspension was prepared by dispersing 1: 1 total cells in a DMEM mixed medium at a ratio of 1 × 10 ⁵ cells / ml. The electrospun nanofibers prepared in Example 1 were placed in a 96-well plate and seeded with the cell dispersion prepared above. Cell-seeded electrospun nanofibers were incubated at 37 占 폚 for 2 hours for cell attachment.

After the ADSC and myocardial cells were adhered to the electrospun nanofibers, HUVEC was dispersed in a mixed medium of EGM-2 Bullekit at a concentration of 1 × 10 ⁵ cells / ml to prepare a HUVEC dispersion. 40 μl of radial nanofibers and fibrinogen hydrogel precursor solution, 10 μl of cell dispersion, and 40 μl of thrombin solution were mixed in a 96-well plate, followed by mixing at 37 ° C. for 30 minutes.

In order to coat the cultured electrospun nanofibers and the fibrin hydrogel complex with the alginate hydrogel, 100 μl of a 2 wt% alginate hydrogel precursor solution was placed in a 96-well plate, The radial nanofibers and the fibrin hydrogel complex were placed and 50 μl of calcium chloride solution was added. Nano cages were prepared containing cells that were complexed with electrospun nanofiber, fibrin hydrogel, and alginate hydrogel in which three cells were co-cultured at 37 ° C for 5 minutes.

Experimental Example 3: Determination of the viability of HUVEC in the fibrin hydrogel

To confirm the viability of the HUVEC in the fibrin hydrogel, HUVEC was dispersed in a mixed medium of EGM-2 Bullekit at a concentration of 1 x 10 ⁵ cells / ml to prepare a HUVEC dispersion. In the absence of nanofibers, fibrinogen 40 μl of the hydrogel precursor solution, 10 μl of the above cell dispersion, and 40 μl of the thrombin solution were mixed well and blended at 37 ° C. for 30 minutes, followed by culture for one week (cultured in EGM-2 Bullekit mixed medium) Cell viability was confirmed. After incubation for one week, cells were stained using Live / Dead cell viability assay and confirmed by fluorescence microscopy. The results are shown in Fig. 7

FIG. 7 shows that HUVEC was dispersed in fibrin hydrogel at a concentration of 1 × 10 ⁵ cells / ml in a fibrin hydrogel without electrospun nanofibers for one week in order to confirm the viability of the HUVEC in the fibrin hydrogel. Survival was assessed by Live / Dead cell viability assay. Living cells are green, dead cells are red.

According to the results, it was confirmed that HUVEC in the fibrin hydrogel exhibits a high survival rate.

Experimental Example 4: Examination of structure of nanocage containing cells

In order to confirm the cross-section of the nanocage not containing cells, the fibrin hydrogel was coated with alginate hydrogel in the same manner as in Example 4 except that only the electrospun nanofibers were not used, and then the mixture was mixed with EGM-2 Bullekit mixed medium After 1, 4, and 7 days of incubation, the cross sections of the two hydrogels were examined through an optical microscope. The results are shown in Fig.

FIG. 8 is a graph showing the results of the comparison between the primary hydrogel and the secondary hydrogel in view of the composite appearance. The fibrin hydrogel containing HUVEC without the electrospun nanofibers was coated with alginate hydrogel, , And a photograph of a cross section of two hydrogels through an optical microscope after culturing for 7 days. On the border, the inside shows the fibrin hydrogel in which the cells are present, and the outside shows the cell-free alginate hydrogel.

According to FIG. 8, it was found that HUVEC was well encapsulated only in the fibrin hydrogel.

Experimental Example 5: Determination of vascular capacity of fibrin hydrogel containing cells

In order to confirm the vascularity in fibrin hydrogel, HUVEC was dispersed in a mixed medium of EGM-2 Bullekit at a concentration of 1 × 10 ⁵ cells / ml to prepare a HUVEC dispersion. 40 μl of the fibrinogen hydrogel precursor solution, μl, and thrombin solution (40 μl) were mixed in a 96-well plate, followed by crosslinking at 37 ° C for 30 minutes. The fibrin hydrogel containing the cells prepared by the above method was incubated in a mixed medium of EGM-2 Bullekit for 1 week, and the angiogenesis of HUVEC was confirmed by an optical microscope. The results are shown in Fig.

FIG. 9 is a graph showing the results of measurement of HUVEC-containing fibrin hydrogel without radiofrequency nanofibers in order to confirm the suitability of fibrin hydrogel for angiogenesis. After the fibrin hydrogel was prepared at 1 day, 4 days, and 7 days, The angiogenesis process was observed.

Experimental Example 6: Test for confirming cell activity in a nanocage containing cells

In order to confirm the cell activity of ADSCs in the nanocage, ADSCs were dispersed in a DMEM mixed medium at a ratio of 1 × 10 ⁵ cells / ml to 1 × 10 ⁵ cells / ml to prepare a cell dispersion. The electrospun fiber prepared in Example 1 was placed in a 96-well plate and seeded with the cell dispersion prepared above. Cell-seeded electrospun nanofibers were incubated at 37 占 폚 for 2 hours for cell attachment. Thereafter, a fibrin hydrogel composite was prepared in the same manner as in Example 2, and a nanocage was prepared in the same manner as in Example 3. [ The nano cage containing the ADSCs thus prepared was cultured in a mixed medium of EGM-2 Bullekit for 1 week and the activity of ADSC was measured using CCK-8 reagent.

Separately, in order to confirm the cell activity of HUVEC in the nanocage, HUVEC was dispersed in a mixed medium of EGM-2 Bullekit at a concentration of 1 × 10 ⁵ cells / ml to prepare a HUVEC dispersion, and the cells prepared in Example 1 40 μl of the radioactive nanofibers and the fibrinogen hydrogel precursor solution, 10 μl of the cell dispersion and 40 μl of the thrombin solution were mixed in a 96-well plate, followed by mixing at 37 ° C. for 30 minutes. Thereafter, a nanocage was prepared according to the method in Example 3. [ The nano-cage containing the HUVEC thus prepared was cultured in a mixed medium of EGM-2 Bullekit for 1 week and the activity of ADSC was measured using CCK-8 reagent. The results are shown in Fig.

10 shows the results of measuring the cell activity by incubating HUVEC and ADSC on fibrin hydrogel and electrospun nanofibers for 1 week in order to observe cell activity in the nanocage. As a result, The activity of the cells was confirmed to be maintained.

Claims (21)

Nanofibers for culturing tissue regenerating cells;
A porous first hydrogel layer encapsulating the nanofibers in a first hydrogel, wherein the porous first hydrogel layer has a pore size of 1 um to 100 um for vascular cell culture; And
A composite of nanofibers and hydrogels comprising a porous second hydrogel layer having a pore size of 1 nm to 10 nm for blocking cell adhesion incubation, encapsulating said first hydrogel layer in a second hydrogel. The composite according to claim 1, wherein the nanofiber is an electrospun nanofiber. The method of claim 2, wherein the nanofiber is selected from the group consisting of chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), polylactic acid (3-hydroxybutyrate) -co- (3-hydroxyvalerate) (PHBV), polydioxanthone (PLGA), glycolic acid (PGA), poly PDO), poly [(L-lactide) -co- (caprolactone)], poly (ester urethane) (PEUU), poly [L- (PV), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polystyrene (PS), polyaniline (PAN) Wherein the composition is an electrospun nanofiber fabricated from a biocompatible polymer selected from the group consisting of combinations. delete The complex of claim 1, wherein the first hydrogel is a hydrogel of a biocompatible polymer selected from the group consisting of collagen, chitosan, fibrin, copolymers thereof, and any combination thereof. delete The method of claim 1, wherein the second hydrogel is selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyacrylic acid (PAA), polyvinyl alcohol (PVA) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), gelatin, alginic acid, carrageenan, chitosan, hydroxyalkyl A hydrogel of a biocompatible polymer selected from the group consisting of cellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylate, polyvinyl chloride, maleic anhydride / vinyl ether, Gel. The composite according to claim 1, wherein the strength is from 0.1 to 0.5 MPa. The composite according to claim 1, comprising a tissue regenerating cell adhered to the nanofiber. [Claim 11] The method according to claim 9, wherein the tissue regeneration cell is selected from the group consisting of myocardial cells, fibroblasts, vascular endothelial cells, smooth muscle cells, neurons, chondrocytes, bone cells, skin cells, Schwann cells and stem cells . 10. The composite of claim 9, wherein the first hydrogel layer comprises a cell, a biomaterial, or any combination thereof. 12. The complex of claim 11, wherein said first hydrogel layer comprises human umbilical vein endothelial cells (HUVEC). The method according to claim 1,
A fibroblast containing stem cells and myocardial cells adhered to nanofibers,
Wherein the first hydrogel layer contains HUVEC. A tissue regeneration scaffold comprising a complex according to any one of claims 1-3, 5, and 7-13. 15. The scaffold of claim 14, which is for cardiovascular tissue regeneration. 15. The scaffold of claim 14, wherein the scaffold is for wound healing. Forming a first hydrogel layer for encapsulating the nanofibers by adding a nanofiber into a mold, adding a first hydrogel precursor solution, and then performing a crosslinking reaction; And
And forming a second hydrogel layer for encapsulating the first hydrogel layer by adding a second hydrogel precursor solution to the first hydrogel layer and then performing a crosslinking reaction. ≪ RTI ID = 0.0 > 11. < / RTI > [Claim 18] The method according to claim 17, wherein the first hydrogel precursor is fibrinogen, and the crosslinking reaction is a crosslinking reaction using thrombin as a crosslinking agent. [Claim 19] The method according to claim 17, wherein the second hydrogel precursor is an alginate aqueous solution, and the crosslinking reaction is carried out using calcium chloride as a crosslinking agent. [17] The method of claim 17, wherein the nanofiber is seeded on a nanofiber, followed by culturing. 21. The method according to claim 20, wherein the cell dispersion is a mixed dispersion of cardiac muscle cells and stem cells, and the first hydrogel precursor solution contains HUVEC.

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