WO2020204230A1 - Composite of nanofiber and hydrogel, and scaffold for tissue regeneration comprising same - Google Patents

Abstract

One aspect of the present invention provides a composite of a nanofiber and a hydrogel, which comprises a nanofiber, a first hydrogel layer encapsulating the nanofiber in a first hydrogel, and a second hydrogel layer encapsulating the first hydrogel layer in a second hydrogel; a scaffold for tissue regeneration comprising the composite; and a method for producing same.

Description

Composite of nanofiber and hydrogel and scaffold for tissue regeneration including the same

The present invention relates to a composite of nanofibers and hydrogel and a scaffold for tissue regeneration including the same, and more specifically, a composite of nanofibers and hydrogel that can be effectively used for regeneration of biological tissues, particularly myocardial tissues, and It relates to a scaffold for tissue regeneration, and a method of manufacturing the same.

Cardiovascular diseases such as coronary artery disease, arrhythmia, and heart failure have rapidly increased due to westernized diet, lack of exercise, and aging. Death from working with heart disease now has the second highest mortality rate among major causes of death. Cardiovascular for the fundamental treatment of cardiovascular disease. Tissue regeneration is very important.

Diseases caused by cardiovascular and myocardial tissue damage are difficult to treat and regeneration is difficult, so in the past, 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 heavy burden on patients. In addition, the cell transplantation treatment method is a method of allografting cardiomyocytes into damaged tissues, but it is still impossible to proliferate as many cardiomyocytes as necessary at an appropriate time, and thus, there is a need to develop a technology to replace them.

In order to solve the above problems, a method of regenerating tissues by creating artificial tissues outside the body using cells and effectively transplanting them into the body has recently been in the spotlight, and research on this has been actively conducted.

Patent Document 1 discloses a method of encapsulating human pancreatic cells and PDX1 (duodenal homeobox gene 1) positive human pancreatic progenitor cells using a device based on a polyethylene glycol hydrogel, a biocompatible polymer. According to this, there is an advantage that the encapsulated progenitor cells are well differentiated, but there is a disadvantage in that physical properties such as biodegradability of the hydrogel cannot be easily adjusted because differentiation is controlled using a differentiation medium.

Patent Document 2 discloses a method of encapsulating PDX1-positive human pancreatic progenitor cells by fabricating a three-dimensional cell encapsulation device using PDMS (polydimethylsiloxane). This method is effective for cell differentiation, but due to the nature of PDMS, a material constituting the device, cell adhesion is low and modification is difficult, so it is difficult to control physical properties.

In order to improve such problems, a method of encapsulating cells by producing a composite of nanofibers and hydrogel by electrospinning has been developed. Patent Document 3 devised a hydrogel composite of nanofibers and 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 nature of alginate hydrogel, there is a problem in that it does not proliferate smoothly with respect to adherent and proliferating cells, and thus, it is difficult to generate very important blood vessels during tissue regeneration.

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

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

Another object of the present invention is to provide a method for preparing the composite.

One aspect of the present invention

Nanofibers;

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

It provides a composite of nanofibers and a hydrogel, including a second hydrogel layer, encapsulating the first hydrogel layer in a second hydrogel.

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

Another aspect of the present invention

Forming a first hydrogel layer encapsulating the nanofibers by putting the nanofibers into a frame, adding a first hydrogel precursor solution, and then crosslinking the nanofibers; And

A composite of nanofibers and hydrogel according to the above aspect, comprising the step of forming a second hydrogel layer encapsulating the first hydrogel layer by adding a second hydrogel precursor solution to the first hydrogel layer and then crosslinking reaction It provides a method of manufacturing.

The composite of nanofibers and hydrogel according to an aspect of the present invention introduces a first hydrogel layer capable of attaching and cultivating cells to the nanofibers, thereby culturing cells for tissue regeneration on the nanofibers, and the first hydrogel layer It is possible to cultivate vascular cells essential for tissue regeneration. Moreover, the complex includes a second hydrogel layer at the outermost surface of which cells are difficult to adhere to, so that when the complex is transplanted into a living body for tissue regeneration as a scaffold for tissue regeneration, fibroblasts in the living body and It can block the fibrosis process by preventing the adhesion of collagen, etc., enabling smooth transplantation. Therefore, the complex according to an aspect of the present invention can sufficiently form blood vessels, which are essential for tissue regeneration, and avoid the rejection reaction of the living body such as fibrosis that may occur during the scaffold transplantation process, so that more effective tissue regeneration It can be used as a scaffold for use, especially as a scaffold for cardiovascular tissue regeneration.

1 is a schematic diagram of a scaffold for tissue regeneration according to an embodiment of the present invention, comprising electrospun nanofibers, a fibrin hydrogel layer encapsulating the nanofibers, and an alginate hydrogel layer encapsulating the fibrin hydrogel layer. , In the alginate hydrogel layer, biomaterials may be released and exchanged, and protein and cell invasion from the outside may be prevented or controlled, and co-culture of cells may be performed on the electrospun nanofibers, and the fibrin hydrogel The creation of vascular tissue is possible.

Figure 2 is the present, including the process of manufacturing a nanofiber scaffold by electrospinning (step 1), disinfecting the scaffold (step 2), encapsulating fibrin hydrogel (step 3), and encapsulating alginate hydrogel (step 4). It is a schematic diagram showing the manufacturing process of the composite according to one embodiment of the invention.

3 shows the manufacturing process (a,b) of nanofibers by electrospinning, and the shape (c,d) of the electrospun nanofiber scaffold as viewed through a scanning electron microscope.

4 is a composite of each fibrin hydrogel (a) prepared in Examples 1 to 3, a composite of electrospun nanofibers and fibrin hydrogel (b), an electrospun nanofiber, fibrin hydrogel, and a composite of alginate hydrogel ( This is a photograph of the result of observing c) with the naked eye.

5 is a composite of electrospun nanofibers (Fibrin), electrospun nanofibers and fibrin hydrogel (Fibrin-PCL) prepared in Examples 1, 2 and 3, and electrospun nanofibers, fibrin hydrogel, and alginate hydro A graph showing the results of measuring the mechanical strength of the gel composite (Fibrin-PCL-alginate) is shown.

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

Figure 7 is to confirm the viability of HUVECs inside the fibrin hydrogel, without electrospun nanofibers, HUVECs were dispersed in the fibrin hydrogel at a ratio of 1 Х 10 ⁵ cells/ml, cultured for a week, and This is a picture of the results of confirming the viability by staining with the Live/Dead cell viability assay.

Figure 8 is, in order to confirm the appearance of the composite of the first hydrogel and the second hydrogel, after coating the HUVEC-containing fibrin hydrogel with alginate hydrogel without electrospun nanofibers, 1, 4, in EGM-2 Bullekit mixed medium. And after incubation for 7 days, the cross section of the two hydrogels was taken through an optical microscope.

9 is a fibrin hydrogel containing HUVEC without electrospun nanofibers to confirm the suitability of angiogenesis of a fibrin hydrogel, and then, angiogenesis of HUVEC through an optical microscope at 1, 4 and 7 days. This is a picture of the result of observing the process.

10 is a graph showing the results of measuring the activity of cells by culturing HUVEC and ADSC in fibrin hydrogel and electrospun nanofibers for 1 week, respectively, in order to observe the activity of cells inside the nanocage according to an 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, unless otherwise defined, are used in the same sense as those of ordinary skill in the art generally understand in the related field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention. In addition, the numerical values described herein are considered to include the meaning of "about" even if not specified. In the present specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated. The contents of all publications referred to herein by reference are incorporated herein by reference in their entirety.

The present inventors have carefully studied not only the cultivation of cells for tissue regeneration, but also the scaffold that is advantageous for culturing vascular cells that can form vascular tissue essential for tissue regeneration, and as a result, nanofibers, the primary encapsulating the nanofibers A composite of nanofibers and hydrogels, in which a hydrogel layer and a second hydrogel layer encapsulating the second hydrogel layer were introduced, was conceived.

In one aspect the present invention,

Nanofibers;

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

It provides a composite of nanofibers and a hydrogel, including a second hydrogel layer, encapsulating the first hydrogel layer in a second hydrogel.

In the present specification, the composite of nanofibers and hydrogel according to an aspect of the present invention is also referred to as a “nano cage”.

Among the complexes, the nanofibers are located inside the complex, and when the complex is used as a scaffold for tissue regeneration, it is used for culturing cells for tissue regeneration, for example, co-culture of stem cells and cardiomyocytes. It is a part.

The nanofiber is not particularly limited as long as it is a nanofiber in which cells for tissue regeneration can be cultured, but in one embodiment, the nanofiber is an electrospun nanofiber manufactured by electrospinning. The electrospun nanofibers may be any electrospun nanofibers conventionally known to be suitable for culturing living cells.

The electrospun nanofibers are an aggregate of fibers having a diameter of several tens of nanometers to several micrometers, and the structure includes a plurality of pores of various sizes, and forms a three-dimensional structure capable of attaching cells. I can. In addition, these voids have a predetermined shape, size and volume. The diameter of the electrospun nanofibers can be changed by adjusting conditions such as the concentration of the polymer solution used as the material of the electrospun nanofibers, the flow rate of the electrospinning, and the voltage, and can also be changed by adding a salt to the polymer solution.

In one embodiment, the size of the pores of the electrospun nanofibers may be adjusted to 20 to 100 μm. Since most of the cells are fixed on the scaffold and exist at 10 to 15 µm before transformation occurs, cells for tissue regeneration enter the pores within the above size range and are fixed inside the scaffold, making it suitable for growth and differentiation. have.

The electrospun nanofibers may be nanofibers manufactured by a known electrospinning method. The material used to produce the nanofibers may be any material suitable for culturing cells for tissue regeneration and capable of producing nanofibers. The scaffold formed of nanofibers may be subjected to additional modifications to ensure that the biomaterial is well fixed to the nanofibers. For example, for the additional modification, an oxygen plasma treatment, a radiation grafting method, or a self-assembly monolayer (SAM) method may be used. In the case of oxygen plasma treatment, nanofibers with high hydrophobicity are used to increase hydrophilicity. By using a benzophenone and azide material, for example, by UV irradiation on a nanofiber scaffold through a radiation grafting method, reactivity can be given to transform the surface of the nanofiber into a desired material. The SAM method uses a spontaneous reaction between a surface where a hydroxy functional group exists and silane (Silane) to fix a portion bonded to the silane to the surface. In the radiation grafting or SAM method, in general, N-hydroxysuccinimide (NHS), a substance that chemically binds to a protein, is combined. Since the additional modification to the scaffold formed of such nanofibers allows the biomaterial to be fixed well regardless of the type of the nanofiber-generating material, there is no limitation on the type of the nanofiber-generating material.

The type of polymer forming the electrospun nanofibers is not particularly limited as long as it does not inhibit the culture of cells for tissue regeneration, and various natural polymers and synthetic polymers may be used. A polymer material that does not have biodegradable properties in terms of cell adhesion may be used, but preferably a polymer material having biodegradation properties in terms of drug delivery, cell transplantation and cell therapy may be used. Such biodegradable polymers are, for example, chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), polylactic acid (PLA), and polyglycol. Acid (PGA), poly[(lactic-co-(glycolic acid))(PLGA), poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (PHBV), polydioxanone (PDO) ), poly[(L-lactide)-co-(caprolactone)], poly(ester urethane)(PEUU), poly[(L-lactide)-co-(D-lactide)], poly[ethylene -co-(vinyl alcohol)](PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyaniline (PAN), and any combination thereof It can be selected from the group consisting of.

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

If necessary, the nanofibers may be coated with fibronectin for smoother adhesion of cells. In addition, in terms of drug delivery, growth factors used to help proliferation and differentiation of cells can be introduced into electrospun nanofibers. To this end, in the electrospinning process, the polymer solution may be mixed and injected into the fiber, or may be physically/chemically attached to the outside of the fiber to be introduced.

The electrospun nanofibers are prepared by electrospinning a polymer solution, and may be prepared using a method commonly used in the art.

A polymer solution for electrospinning may be prepared by selecting a suitable solvent capable of dissolving the polymers as listed above for the production of electrospun nanofibers. Such a solvent is not particularly limited as long as the above conditions are satisfied, but for example, chloroform, trifluoroethanol (2,2,2-trifluoroethanol, TFE), tetrahydrofuran (THF), dimethylform It may be selected from aldehydes (Dimethylformaldehyde, DMF), and any combination thereof, and may be appropriately selected by a person skilled in the art in consideration of factors such as viscosity and dielectric constant of the solution.

In the electrospinning method for producing 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 surface tension of the polymer solution is exceeded by the mutual repulsion between the same charges. As the solvent evaporates, the fibers are charged with the opposite charge transferred to the solution or collected into a grounded substrate. The diameter of the fiber can be adjusted by controlling the concentration, flow rate, voltage, etc. of the solution in which the polymer is dissolved using the solvent, and the density of the fiber can be controlled by controlling the electrospinning time and height of the nozzle and the substrate.

The solvent used in the polymer solution for preparing the electrospinning nanofibers may have toxicity that is not suitable for culturing cells for tissue regeneration, and is not completely evaporated during the electrospinning process and remains in the obtained nanofibers, causing cytotoxicity. There is concern. Therefore, in order to use the complex as a scaffold for tissue engineering, 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 C 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 with the first hydrogel and then again encapsulated with the second hydrogel, thereby forming the nanofiber and hydrogel composite.

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 with the first hydrogel by, for example, putting nanofibers in a hydrogel precursor solution and crosslinking the hydrogel precursor solution to form a hydrogel. In addition, the nanofibers encapsulated with the first hydrogel may be encapsulated with the second hydrogel in the same manner.

The first hydrogel is a hydrogel suitable for culturing vascular cells required for blood vessel formation due to its high cell adhesion, and the second hydrogel has low cell adhesion and prevents adhesion proliferation when the complex is transplanted into a living body as a scaffold. It is a hydrogel that can prevent external invasion of cells. That is, the first hydrogel has excellent adhesion of vascular endothelial cells and plays a role that can greatly aid in proliferation and angiogenesis, and the second hydrogel has a significantly smaller pore size than the first hydrogel, and adheres to cells. The ability to encapsulate cells is poor, but it is not helpful for the proliferation of adherent-proliferating cells, but has the advantage of preventing external invasion of proteins and cells during the proliferation of non-adherent proliferating cells and transplantation into the body.

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

Because hydrogels have a three-dimensional structure like electrospun nanofibers, they help in three-dimensional cultivation of cells, but can show a large difference in the adhesion of cells depending on the properties of the polymers that make up the hydrogel. For example, macromolecules such as collagen exhibit good properties for cell adhesion in the form of a hydrogel, but polymers such as PEG and alginate have great restrictions on cell adhesion when produced as a hydrogel. Suitable. Therefore, the first hydrogel and the second hydrogel can be prepared by appropriately selecting a polymer. In addition, the cell adhesion may vary depending on the conditions for producing the hydrogel. Therefore, the cell adhesion capability of the hydrogel may be adjusted by controlling the porosity according to the production conditions, and according to methods known in the art. Accordingly, a person skilled in the art can properly manufacture.

The first hydrogel may be formed of a biocompatible polymer suitable for culturing vascular cells required for angiogenesis, for example, selected from the group consisting of collagen, chitosan, fibrin, copolymers thereof, and any combination thereof. It may be a 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 vascular cells, thereby enabling the formation of blood vessels essential for tissue regeneration.

The second hydrogel may be formed of a biocompatible polymer while being able to block adhesion and proliferation of cells. For example, polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), Polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycapro Lactone (PCL), gelatin, alginic acid, carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylic acetate, polyvinyl chloride, maleic anhydride/vinyl ether, And it may be a hydrogel of a biocompatible polymer selected from the group consisting of any combination thereof, but is not limited thereto.

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

Since the composite of the nanofibers and the hydrogel further includes a secondary hydrogel layer, when the composite is implanted as a scaffold, there is an advantage that it is possible to block rejection reactions in the body such as fibrosis. When transplanting into a living body using only a scaffold consisting of nanofibers or a scaffold consisting of nanofibers and a porous first hydrogel, fibrosis occurs and the effect of transplantation of external tissues is very poor. In the case of passivation by encapsulating the hydrogel, excellent effects can be obtained for in vivo transplantation.

The first hydrogel layer and the second hydrogel layer may form a hydrogel layer by adding a hydrogel precursor solution and performing a crosslinking reaction. Polymers forming hydrogels have various crosslinking methods according to their respective characteristics. For example, photo-crosslinking by ultraviolet (UV) irradiation, crosslinking using a reactive crosslinker, etc. are mentioned. The crosslinked hydrogel using this method is not easily decomposed and crosslinked. In the process, a patterning technique can be applied by crosslinking the hydrogel only in a specific area using a mask. In addition, the thermal-crosslinking method, which crosslinks by temperature, is crosslinked at a specific temperature or higher to form a hydrogel, and can be applied to cell release and drug delivery through temperature change. In addition, crosslinking methods using specific protein reactions and reactions between ions and polymers have the advantage of being able to crosslink hydrogels using well-known reactions between proteins, and to easily produce hydrogels.

In one embodiment, when fibrin hydrogel is included as the first hydrogel, fibrin hydrogel may be formed by using fiprinogen as a hydrogel precursor solution and adding thrombin as a crosslinking agent. In addition, when the alginate hydrogel is included as the second hydrogel, the alginate hydrogel may be formed by using the alginate salt solution as a precursor solution such as calcium chloride.

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 drugs from the inside. The hydrogel may be capable of sustained drug release. In order to introduce a drug into the hydrogel, if the drug is added before the crosslinking reaction for the hydrogel form and then crosslinked, the drug can be incorporated into the hydrogel, and the drug incorporated from the hydrogel can be continuously delivered. In addition, as another method for introducing a drug into the hydrogel, the drug may be incorporated into the hydrogel from the medium by adding the drug to an external medium in the cell culture process using the hydrogel.

In one embodiment, VEGF (Vascular endothelial growth factor) or S1P (Sphingosin-1-phosphate), which is a growth factor helpful for angiogenesis, may be mixed in a medium and transferred into the hydrogel, and the growth factor is the first hydro It can promote blood vessel formation in the gel layer.

The composite of the nanofibers and the hydrogel may have sufficient strength such that it is not easily damaged during implantation in vivo for tissue regeneration. The composite may have better strength due to the fact that it includes a first hydrogel layer and a second hydrogel layer encapsulating nanofibers, the strength may vary depending on the degree of crosslinking of the hydrogel, the thickness of the nanofibers, the thickness of the hydrogel, etc. It can be adjusted according to a known method by a person skilled in the art. Accordingly, if the complex has a sufficient strength, a scaffold for tissue regeneration can be produced similar to the physical strength of the extracellular matrix of each tissue to be regenerated. If the physical strength is similar, the tissue of the cells in the scaffold It has the advantage that specific specificity can be easily assigned.

In one embodiment, the composite may have a strength of 0.1 to 0.5 MPa. When the intensity is higher than the above intensity, blood vessel regeneration may not be performed smoothly, and when it is low, it may be difficult to control the differentiation of stem cells.

The complex may include cells for tissue regeneration adhered and cultured on the nanofibers to be used as a scaffold for tissue regeneration.

Cells for tissue regeneration may be used for any desired tissue regeneration, for example, cardiomyocytes, smooth muscle cells, cartilage cells, bone cells, skin cells, fibroblasts, vascular endothelial cells, neurons, Schwann cells. , Stem cells, and any combination thereof, but is not limited thereto. In one embodiment, cardiomyocytes and stem cells may be co-cultured and introduced on the nanofibers.

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

In one embodiment, the first hydrogel layer may contain human umbilical cord blood vein endothelial cells (HUVEC), and because of containing HUVEC, angiogenesis may be promoted, and thus tissue regeneration may be remarkably advantageous. Additionally, the first hydrogel layer may contain a growth factor, and due to the inclusion of the growth factor, the proliferation of cells adhered to the nanofibers and proliferation of vascular cells in the first hydrogel layer may be promoted. In one embodiment, the growth factor is vascular endothelial growth factor (VEGF).

In one embodiment, the complex contains cardiomyocytes and stem cells adhered and cultured on nanofibers, and the first hydrogel layer contains HUVEC.

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

Details of the scaffold for tissue regeneration may be the description of the composite of nanofibers and hydrogels according to an aspect of the present invention.

In the present specification, the term "scaffold" refers to a structure that serves to provide an environment suitable for attachment, differentiation of cells, and proliferation and differentiation of cells moving from around tissues as one of important basic elements in the field of tissue regeneration engineering.

The tissue regeneration scaffold may determine a tissue capable of regeneration according to the type of cells adhered and cultured on the nanofibers. For example, cardiomyocytes for cardiovascular tissue regeneration, cartilage cells for cartilage tissue regeneration, bone cells for bone tissue regeneration, skin cells for skin tissue regeneration, fibroblasts for wound tissue regeneration, and the like may be used.

In one embodiment, the tissue regeneration scaffold is a cardiovascular tissue regeneration scaffold, and contains adherent-cultured cardiomyocytes or additional stem cells on the nanofibers.

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

Conventionally, when a scaffold containing cells is implanted in vivo, many cases of transplant failure due to fibrosis in vivo have been reported. However, the scaffold for tissue regeneration according to the present invention can block the fibrosis process by blocking the adhesion of fibroblasts and collagen in a living body to the scaffold due to passivation due to the introduction of a secondary hydrogel layer, so that smooth transplantation is possible. .

In addition, the scaffold for tissue regeneration can form vascular tissue by culturing vascular cells in the first hydrogel layer, so that when the scaffold is implanted in vivo, it can stably and effectively regenerate living tissue.

Therefore, the complex according to an aspect of the present invention is capable of sufficiently forming blood vessels essential for tissue regeneration and avoiding the rejection reaction of the living body such as fibrosis that may occur during the scaffold transplantation process. It can be used as a scaffold, in particular a scaffold for cardiovascular tissue regeneration.

In addition, the scaffold for tissue regeneration can enable continuous drug release by incorporating desired drugs into electrospun nanofibers and hydrogels, and scaffolds by incorporating drugs effective in constructing an extracellular matrix (ECM) It can promote the smooth formation of extracellular matrix in surrounding tissues to be transplanted. Due to the inclusion of these drugs, more effective applications are possible in fields such as wound healing.

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

The scaffold for tissue regeneration according to FIG. 1 has electrospun nanofibers, a fibrin hydrogel layer encapsulating the nanofibers, and an alginate hydrogel layer encapsulating the fibrin hydrogel layer. In the alginate hydrogel layer, biomaterials can be released and exchanged, protein and cell invasion from the outside can be prevented or controlled, and co-culture of cells can be performed on the electrospun nanofibers, and blood vessels in the fibrin hydrogel Organizational creation is possible.

In another aspect of the present invention,

Forming a first hydrogel layer encapsulating the nanofibers by putting the nanofibers into a frame, adding a first hydrogel precursor solution, and then crosslinking the nanofibers; And

A nanofiber and hydrogel according to an aspect of the present invention comprising the step of forming a second hydrogel layer encapsulating the first hydrogel layer by adding a second hydrogel precursor solution to the first hydrogel layer and then crosslinking. It provides a method for preparing a gel complex.

Details of the method of manufacturing the composite of the nanofiber and the hydrogel may be applied to the description of the composite of the nanofiber and the hydrogel according to an aspect of the present invention.

In one embodiment, the first hydrogel precursor is fibrinogen, and the crosslinking reaction may be performed by using thrombin as a crosslinking agent. In addition, the second hydrogel precursor is an aqueous alginate solution, and the crosslinking reaction may be performed by using calcium chloride as a crosslinking agent. The aqueous alginate solution is, for example, sodium daily acid.

The nanofibers may be used after seeding the cell dispersion of cells for tissue regeneration as described above on the nanofibers and then culturing them.

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

Fig. 2 is a schematic diagram showing a step-by-step process of a method for preparing a composite of the nanofiber and hydrogel according to an embodiment. According to FIG. 2, preparation of nanofibers by electrospinning (step 1), disinfection of nanofibers (step 2), formation of a fibrin hydrogel layer encapsulating nanofibers (step 3), and encapsulating the fibrin hydrogel layer It includes the process of forming an alginate hydrogel layer (step 4).

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereby.

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

After mixing 0.5 g of polycaprolactone (PCL, Sigma) and 0.5 g of gelatin (Gelatin, Sigma) in 5 ml of trifluoroethanol (TFE, Sigma), completely sealed so that the solvent does not evaporate, and put for 12 hours at 80°C. The gelatin was completely dissolved. After cooling the prepared polymer solution at room temperature, vortexing was performed to thoroughly mix.

For electrospinning, the solution is placed in a syringe and mounted in the electrospinning system, and then the solution is pushed at a constant flow rate of 0.7 ml/hr using a syringe pump, and a cylindrical needle made of metal (Needle). tip). A voltage of 8kV was applied to a cylindrical needle made of metal using a high voltage device (power supply). After grounding the collecting plate to receive the fibers obtained by electrospinning, aluminum foil is laid on it to make a fixed size electrospun nanofiber scaffold with a cover glass of 1.8 cm x 1.8 cm ( A fibrous sheet was obtained through electrospinning for 30 minutes by placing a frame wrapped with aluminum foil on the dust collecting plate. The distance between the cylindrical needle to which the voltage was 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 by the grounded dust collecting plate to form a fiber layer. The resulting electrospun nanofibers were placed in a vacuum at 60° C. for 24 hours to remove residual solvent.

3A and 3B are photographs of the electrospun fiber from which the residual solvent was removed in a vacuum state in the above method using a mobile phone camera, and c and d are the electrospun fibers coated with platinum of 15 mA for 1 minute and then scanning electrons This is a picture taken through a microscope.

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

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

To prepare a fibrin hydrogel precursor solution (i.e., a fibrinogen solution), 0.1 mg of sodium chloride (NaCl) was added to 5 ml of distilled water and dissolved by vortexing for 30 minutes at room temperature. Then, fibrinogen from Human plasma, Sigma) 100 mg was added and dissolved at room temperature for 10 minutes.

In addition, thrombin was used as a crosslinking agent to crosslink fibrinogen with fibrin.To prepare a thrombin solution, 0.2 mmol of calcium chloride (CaCl ₂ ) was added to 5 ml of distilled water and dissolved by vortexing for 30 minutes at room temperature. A fibrin solution was prepared by adding 100 U (Enzyme unit) of thrombin to the solution and dissolving it at room temperature for 10 minutes.

First, put the electrospun nanofibers in a 96 well plate, add the prepared fibrinogen solution to the extent that the electrospun nanofibers are immersed, and put the prepared thrombin solution in an amount having a volume ratio of 1:1 with the fibrinogen solution, and pipetting ( Pipetting) to mix well. The mixed solution was stored for 30 minutes at 37° C. for crosslinking of fibrinogen, and through this, a composite of electrospun nanofibers and fibrin hydrogel was prepared.

Example 3: Electrospun nanofiber, fibrin Hydrogel , And Alginate Hydrogel Composite fabrication

To prepare an alginate hydrogel precursor solution, 0.1 g of sodium alginate (Wako) was added to 5 ml of distilled water, sealed so that distilled water did not evaporate, and completely dissolved at 37° C. for 2 hours. In addition, 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 by the above method was added to a 96 well plate, and a composite of the electrospun nanofibers and fibrin hydrogel prepared in Example 2 was placed therein. Then, in order to form the alginate hydrogel, 50 μl of a calcium chloride solution as a crosslinking agent was put into an aqueous sodium alginate solution and left at room temperature for 5 minutes. As a result, a nanocage, which is a composite of electrospun nanofibers, fibrin hydrogel, and alginate hydrogel, was prepared.

Fibrin hydrogel prepared in Examples 1, 2 and 3 (a), composite of electrospun nanofiber and fibrin hydrogel (b), composite of electrospun nanofiber, fibrin hydrogel, and alginate hydrogel (c) Figure 4 shows a picture of.

Experimental example 1: fibrin Hydrogel , Electrospun nanofibers and fibrin Hydrogel Evaluation of properties of composites and nano cages

After washing the fibrin hydrogel made under the above conditions, the composite of the electrospun fiber and the fibrin hydrogel, and the nanocage with a PBS solution to remove the residual hydrogel precursor, compressive stress and compressive strain were applied. It was placed on an instron (Instron corporation) equipment substrate for measurement.

In measuring the compressive stress and compressive strain of each scaffold, compressive stress and compressive strain were measured by compressing until 80% of the existing height of each scaffold. The compressive stress and compressive strain obtained by the above method were measured using an equation to measure the actual compressive stress and the actual compressive strain, draw a graph, and then measure the slope in a certain section of the graph to calculate the compressive modulus. 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

5 is a composite of electrospun nanofibers (Fibrin), electrospun nanofibers and fibrin hydrogel prepared in Examples 1, 2 and 3 (Fibrin-PCL), and electrospun nanofibers, fibrin hydrogel, and A graph of the mechanical strength of a composite of alginate hydrogel (Fibrin-PCL-alginate) is shown.

Experimental Example 2: Nano cage cross-section confirmation test

The cross section of the prepared nano cage was observed. First, the nanocage was quenched using liquid nitrogen, and then vertically cut, and the vertical section of the nanocage was observed using a cryo-scanning electron microscopy (Cryo-SEM). The results are shown in FIG. 6.

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

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

Example 4: Fabrication of a nanocage containing cells

The medium for culturing adipose-derived mesenchymal stem cells (ADSC) and cardiomyocytes is DMEM (Dulbecco's Modified Eagle's Medium) with 10% Fetal bovine serum (FBS) and 1% Penicillin. A DMEM mixed medium mixed with /streptomycin was used. In addition, as a medium for culturing human umbilical vein endothelial cells (HUVEC), a solution of 2% FBS and 1% Penicillin/streptomycin in EGM-2 Bullekit (Endothelial Cell Growth Medium) was used.

For co-culture of ADSC and cardiomyocytes, a cell dispersion was prepared by dispersing in a DMEM mixed medium at a ratio of 1 to 10 ⁵ cells/ml of total cells at 1:1. The electrospun nanofibers prepared in Example 1 were put into a 96 well plate, and the cell dispersion prepared above was seeded thereon. For cell adhesion, the electrospun nanofibers seeded with cells were cultured at 37° C. for 2 hours.

After ADSC and cardiomyocytes are adhered and cultured on electrospun nanofibers, HUVECs are dispersed in an EGM-2 Bullekit mixed medium at a ratio of 1 Х 10 ⁵ cells/ml to prepare a HUVEC dispersion, and the cells are adhered and cultured. The spun nanofibers and fibrinogen hydrogel precursor solution 40 μl, cell dispersion 10 μl, and thrombin solution 40 μl were added to a 96 well plate, mixed well, and crosslinked at 37° C. for 30 minutes.

Thus, in order to coat the electrospun nanofiber and fibrin hydrogel complex in which the prepared cells are cultured with alginate hydrogel, 100 μl of a 2 wt% alginate hydrogel precursor solution is put into a 96 well plate, and the prepared cells are cultured. After the spinning nanofibers and fibrin hydrogel complex were added, 50 μl of a calcium chloride solution was added. By crosslinking at 37° C. for 5 minutes, a nanocage containing cells, a complex of electrospun nanofibers, fibrin hydrogel, and alginate hydrogel, in which three cells were co-cultured, was prepared.

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

In order to confirm the viability of HUVECs inside the fibrin hydrogel, HUVEC was dispersed in an EGM-2 Bullekit mixed medium at a ratio of 1 Х 10 ⁵ cells/ml to prepare a HUVEC dispersion, and without the presence of nanofibers, fibrinogen 40 μl of the hydrogel precursor solution, 10 μl of the cell dispersion, and 40 μl of the thrombin solution were added to a 96 well plate, mixed well, crosslinked at 37°C for 30 minutes, and incubated for a week (incubated in EGM-2 Bullekit mixed medium). Cell viability was confirmed. After incubation for a week, cells were stained using a Live/Dead cell viability assay, and then confirmed through a fluorescence microscope. The results are shown in FIG. 7

Figure 7 is to confirm the viability of HUVECs inside the fibrin hydrogel, without electrospun nanofibers, HUVECs were dispersed in the fibrin hydrogel at a ratio of 1 Х 10 ⁵ cells/ml, cultured for a week, and This is a picture of the results of confirming the viability by staining with the Live/Dead cell viability assay. Live cells are green and dead cells are red.

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

Experimental Example 4: Structure confirmation test of a nano cage containing cells

In order to confirm the cross section of the nanocage that does not contain cells, the fibrin hydrogel was coated with alginate hydrogel in the same manner as in Example 4 except that there was no electrospun nanofiber, and then EGM-2 Bullekit mixed medium After incubation for 1, 4, and 7 days at, the cross sections of the two hydrogels were confirmed through an optical microscope. The results are shown in FIG. 8.

FIG. 8 is a first hydrogel and a secondary hydrogel after coating the HUVEC-containing fibrin hydrogel with alginate hydrogel without electrospun nanofibers, 1 and 4 in EGM-2 Bullekit mixed medium. , And after incubation for 7 days, the cross section of the two hydrogels was taken through an optical microscope. Based on the boundary, the inner side represents the fibrin hydrogel with cells, and the outer side represents the alginate hydrogel without cells.

According to Figure 8, it was confirmed that HUVEC was well encapsulated only inside the fibrin hydrogel.

Experimental Example 5: Confirmation of angiogenic ability of fibrin hydrogel containing cells

In order to confirm the angiogenic ability in fibrin hydrogel, HUVEC was dispersed in EGM-2 Bullekit mixed medium at a ratio of 1 Х 10 ⁵ cells/ml to prepare a HUVEC dispersion, fibrinogen hydrogel precursor solution 40 μl, cell dispersion 10 μl and 40 μl of thrombin solution were added to a 96 well plate, mixed well, and crosslinked at 37°C for 30 minutes. Fibrin hydrogel containing cells prepared by the above method was cultured in EGM-2 Bullekit mixed medium for 1 week, and then angiogenesis of HUVEC was confirmed through an optical microscope. The results are shown in FIG. 9.

FIG. 9 shows a fibrin hydrogel containing HUVEC without electrospun nanofibers in order to confirm the suitability of angiogenesis of fibrin hydrogel, and then the HUVEC through optical microscopy at the time points of days 1, 4, and 7 The angiogenesis process was observed.

Experimental Example 6: Cell activity confirmation test in a nano-cage containing cells

In order to confirm the cellular activity of ADSC in the nanocage, a cell dispersion was prepared by dispersing ADSC in a DMEM mixed medium at a ratio of 1 Х 10 ⁵ cells/ml in a ratio of 1:1. The electrospun fibers prepared in Example 1 were placed in a 96 well plate, and the cell dispersion prepared above was seeded thereon. For cell adhesion, the electrospun nanofibers seeded with cells were cultured at 37° C. for 2 hours. 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 prepared nano-cage containing ADSC was cultured in EGM-2 Bullekit mixed medium for 1 week, and the activity of ADSC was measured using CCK-8 reagent.

Separately, in order to confirm the cellular activity of HUVEC in the nanocage, HUVEC was dispersed in an EGM-2 Bullekit mixed medium at a ratio of 1 Х 10 ⁵ cells/ml to prepare a HUVEC dispersion, and the cells prepared in Example 1 40 μl of the electrospun nanofibers and fibrinogen hydrogel precursor solution, 10 μl of cell dispersion, and 40 μl of thrombin solution were added to a 96 well plate, mixed well, and crosslinked for 30 minutes at 37°C. After that, a nanocage was manufactured according to the method in Example 3. The nano-cage containing HUVEC thus prepared was cultured in EGM-2 Bullekit mixed medium for 1 week, and the activity of ADSC was measured using CCK-8 reagent. The results are shown in FIG. 10.

10 is a result of measuring the activity of cells by culturing HUVEC and ADSC in fibrin hydrogel and electrospun nanofibers for 1 week, respectively, to observe the activity of cells inside the nanocage.According to this result, culture for 1 week It was also confirmed that the cell activity is maintained.

Claims (21)

Nanofibers; A first hydrogel layer encapsulating the nanofibers in the first hydrogel; And A composite of nanofibers and hydrogel, including a second hydrogel layer, encapsulating the first hydrogel layer in the second hydrogel. The composite of claim 1, wherein the nanofibers are electrospun nanofibers. The method of claim 2, wherein the nanofiber is chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), polylactic acid (PLA), poly Glycolic acid (PGA), poly[(lactic-co-(glycolic acid))(PLGA), poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (PHBV), polydioxanone ( PDO), poly[(L-lactide)-co-(caprolactone)], poly(ester urethane)(PEUU), poly[(L-lactide)-co-(D-lactide)], poly[ Ethylene-co-(vinyl alcohol)](PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyaniline (PAN), and any of these A composite that is an electrospun nanofiber made of a biocompatible polymer selected from the group consisting of a combination. The composite of claim 1, wherein the first hydrogel is a porous hydrogel having pores of 1 um-100 um. The composite 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. The composite of claim 1, wherein the second hydrogel is a porous hydrogel having pores of 1 nm to 10 nm. The method according to claim 1, wherein the second hydrogel is polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N -Isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), gelatin, alginic acid, carrageenan, chitosan, hydroxyalkyl Hydro of a biocompatible polymer selected from the group consisting of cellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylic acetate, polyvinyl chloride, maleic anhydride/vinyl ether, and any combination thereof A complex that is a gel. The composite according to claim 1, wherein the strength is 0.1 to 0.5 MPa. The composite of claim 1, comprising cells for tissue regeneration adhered and cultured on nanofibers. The complex according to claim 9, wherein the tissue regeneration cells are selected from the group consisting of cardiomyocytes, fibroblasts, vascular endothelial cells, smooth muscle cells, neurons, chondrocytes, bone cells, skin cells, Schwann cells, and stem cells. . The complex of claim 9, wherein the first hydrogel layer contains cells, biomaterials, or any combination thereof. The complex of claim 11, wherein the first hydrogel layer contains human umbilical cord blood vein endothelial cells (HUVEC). The method according to claim 1, It contains cardiomyocytes and stem cells adhered and cultured on nanofibers, The complex of the first hydrogel layer containing HUVEC. A scaffold for tissue regeneration comprising the complex according to any one of claims 1 to 13. The scaffold according to claim 14, which is for cardiovascular tissue regeneration. The scaffold according to claim 14, which is for wound treatment. Forming a first hydrogel layer encapsulating the nanofibers by putting the nanofibers into a frame, adding a first hydrogel precursor solution, and then crosslinking the nanofibers; And The composite according to any one of claims 1 to 13, comprising the step of forming a second hydrogel layer encapsulating the first hydrogel layer by adding a second hydrogel precursor solution to the first hydrogel layer and then performing a crosslinking reaction. Manufacturing method. The method of claim 17, wherein the first hydrogel precursor is fibrinogen, and the crosslinking reaction is performed by crosslinking using thrombin as a crosslinking agent. The method according to claim 17, wherein the second hydrogel precursor is an alginate aqueous solution, and the crosslinking reaction is performed by crosslinking using calcium chloride as a crosslinking agent. The method of claim 17, wherein the nanofibers are used after seeding the cell dispersion on the nanofibers and culturing them. The method of claim 20, wherein the cell dispersion is a mixed dispersion of cardiomyocytes and stem cells, and the first hydrogel precursor solution contains HUVEC.

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