KR20190018350A - Nanofibril-reinforced polymer hydrogel complex and preparation method thereof - Google Patents

Abstract

Provided are a nanofibril reinforced polymer hydrogel composite which is able to be used as a cell culture support body and capable of improving the proliferation and survivability of cells, and a manufacturing method thereof. The method includes: a step of making nanofibers by electrically spinning a hydrophobic polymer solution; a step of making nanofibril by hydrolyzing the nanofibers; a step of mixing the nanofibril with the hydrophobic polymer solution; and a step of forming a cross-linked bond of the nanofibril.

Description

[0001] The present invention relates to nanofibril-reinforced polymer hydrate gel complexes and nanofiber-reinforced polymer hydrogel complexes,

More particularly, the present invention relates to a nanofibril-reinforced polymer hydrate gel composite prepared from a hydrophobic polymer and a hydrophilic polymer, and to a nano-fibril-reinforced polymer hydrate gel composite prepared thereby.

Tissue engineering is the application of fundamental concepts and techniques of life sciences and engineering to understand the relationship between structure and function of living tissue, and then to make a substitute for living tissue and transplant it back into the body. Maintenance, enhancement or restoration. In such tissue engineering, tissues of humans or animals are collected, cells are separated from the tissues and cultured on a cell culture support (scaffold) to prepare a cell-support complex, and then the prepared cell- The basic principle is to transplant in.

Tissue engineering techniques have been applied to almost all organs of human body such as artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessel and artificial muscle. In order to optimize the regeneration of such living tissues and organs, basically, Should be provided. The basic requirements of an ideal support are largely non-toxic, mechanical properties and porosity. In addition, biomimetic 3D scaffolds, in particular, can allow cells to behave in a manner similar to in vivo .

Patent Document 1 discloses a hyaluronic acid-based hydrogel complex which can be widely applied to various biotechnological fields such as regenerative medicine, tissue engineering, stem cell culture and differentiation, drug delivery, and the like. However, such hydrophilic hydrogels including hyaluronic acid hydrogels have been insufficient in terms of cell adhesion rate. In addition, cell culture supports prepared from other biodegradable polymers did not show satisfactory cell survival rate, proliferation rate, and adhesion rate.

Therefore, it is necessary to study cell culture supports capable of enhancing cell survival rate, proliferation rate and adhesion rate, and capable of three - dimensional cell culture at the same time as excellent physical properties.

Korean Patent No. 10-1726126

The present invention provides a method for producing a nano-fibril-reinforced polymer hydrate gel composite.

The present invention also provides a nanofibril-reinforced polymer-hydrated gel composite produced by the above-described method.

One aspect of the present invention is

Preparing nanofibers by electrospinning the hydrophobic polymer solution;

Hydrolyzing the nanofibers to produce nanofibrils;

Adding and mixing the nanofibrils to a hydrophilic polymer solution; And

And forming a cross-linking of the nanofibrils. The present invention also provides a method for producing a nanofiber-reinforced polymer-hydrated gel.

In another aspect of the present invention,

The nano-fibril-reinforced polymer-hydrated gel composite produced by the above-described method is provided.

The nano-fibril-enhanced polymeric hydrate gel complex according to one embodiment of the present invention can improve the survival rate, proliferation rate, and spreading effect of the cells, thereby providing an effective three-dimensional cell culture support.

FIG. 1 is a schematic view showing a method of manufacturing a nano-fibril-reinforced polymer-hydrogel composite according to an embodiment.
Figure 2 (a) shows a digital image of a nanofiber-reinforced polymeric hyd ... 2 (B) shows a scanning electron microscope photograph.
Figure 3 (A) shows the modulus of elasticity before culturing the hydrogel in nanofibrils. Figure 3 (B) shows the elastic modulus after incubation of the hydrogel in the nanofibrils for 14 days. 3 (C) shows the result of measuring the weight loss rate (%) of the nanofiber-reinforced polymer-hydrated gel complex. In Figs. 3 (a) to 3 (c), the values obtained by measuring the elastic modulus or the inorganic loss ratio of the hydrogel containing nanofibrils at the ratios of 0, 1, 5, 10, 15 and 20% Green, yellow, blue and purple.
4 shows the survival rate (%) of the cells when the cells were cultured in the nano-fibril-reinforced polymer hydrogel complex for 14 days.
5 (a) (a) to (f) show the result of confocal laser microscopy of cells in a hydrogel when nano-fibrils were contained at a ratio of 0, 1, 5, 10, 15 and 20% . FIG. 5 (B) shows the result of measuring the degree of cell stretching in the nanofibril-reinforced polymer hydrate gel complex. 5 (C) shows the results of measurement of the circumferential length (占 퐉) of the cells in the nanofiber-reinforced polymer hydrate gel complex.
FIG. 6 shows the results of RT-PCR measurement of the gene expression of cells in the nano-fibril. ((A) to (f)): At the ratio of 0, 1, 5, 10, Fibril-containing hydrogels).

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.

The present invention, in one aspect,

Preparing nanofibers by electrospinning the hydrophobic polymer solution;

Hydrolyzing the nanofibers to produce nanofibrils;

Adding and mixing the nanofibrils to a hydrophilic polymer solution; And

And forming a cross-linking of the nanofibrils. The present invention also provides a method for producing a nanofiber-reinforced polymer-hydrated gel.

The term " polymer hydrogel " refers to a three-dimensional network structure formed by crosslinking a biocompatible polymer with a covalent or non-covalent bond. The polymer hydrogel is not dissolved in the aqueous solution, but it can absorb and swell a large amount of water.

The term " nano-fibril-reinforced polymer-hydrate gel complex " refers to a composite material formed by binding nano-fibrils to a polymer hydrogel.

The step of adding the nanofibrils to the hydrophilic polymer solution and mixing the nanofibrils with the nanofibrils may further include adding the target cells to the mixture.

The hydrophobic polymer includes poly (ε-caprolactone) (PCL), poly (lactic acid): PLA, poly (glycolic acid) Glycolic acid copolymer, polylactic-co-glycolic acid (PLGA), and combinations thereof.

 The hydrophilic polymer may be selected from the group consisting of alginate, gelatin, hyaluronic acid, chitosan, pullulan, polyvinyl alcohol (PVA), poly (gamma -glutamic acid) Poly (γ-glutamic acid): γ-PGA), and combinations thereof.

In one embodiment, the hydrophilic polymer is a mixed solution of alginate (Alg) and gelatin (Gel), and the nanofiber-reinforced polymer hydrate gel complex is prepared. In one embodiment, the ratio of the mixed solution of alginate and gelatin may be 3: 1 to 1: 3, for example, 2: 1 to 1: 2.

The step of hydrolyzing the nanofibers may be carried out using a sodium hydroxide (NaOH) solution or hydrochloric acid (HCl) as a catalyst. In one embodiment, the hydrolysis can be performed with 0.05 to 2 M NaOH, such as 1 M NaOH, or 0.5 to 10 M HCl, such as 1 M HCl.

The step of forming the crosslinking of the nanofibrils may be carried out by using any one selected from the group consisting of calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), calcium phosphate (CaP), calcium carbonate (CaCO ₂ ) As a cross-linking agent. In one embodiment, the crosslinking agent may be carried out using a CaCl ₂ solution of 0.05 to 2% as a crosslinking agent.

In one embodiment, the manufacturing method may further include adding a target cell to the mixed solution.

In one embodiment, the step of adding the nanofibrils to the hydrophilic polymer solution and mixing the nanofibrils with the hydrophilic polymer solution may include adding the target cells to the mixture.

In one embodiment, the step of adding the target cell to the mixed solution after the step of forming the crosslinking of the nanofibrils may further include the step of adding the target cell.

In addition, the method may further include a step of culturing the target cells after the step of forming the crosslinking of the nanofibrils.

The subject cell may be an adherent cell that adheres to the bottom or a suspension cell that floats in the medium, preferably an adherent cell. In one embodiment, the cells may grow by adhering to the surrounding environment, such as a support. In one embodiment, the growth of the cells can be measured by a stretch phenomenon in which the cells grow on the support. In one embodiment, the cell may be a fibroblast, a fibroblast, a neuroblast, or a neural cell, for example, a NIH3T3 cell or a HeLa cell. According to another aspect of the present invention, there is provided the nanofibril-reinforced polymer-hydrated gel composite produced by the above production method.

In one embodiment, the nano-fibril-enhanced polymer hydrate gel complex may be used as a scaffold or a support for cell culture.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1. Preparation of nanofiber-reinforced polymer hydrate gel complex

The nano-fibril-reinforced polymer hydrate gel complex was prepared by performing the methods described in Sections 1 and 2 below. FIG. 1 is a schematic view showing a method of manufacturing a nano-fibril-reinforced polymer-hydrogel composite according to an embodiment.

1. Preparation of polycaprolactone nanofibrils

A polycaprolactone solution was prepared by dissolving 2.5 g of poly (ε-caprolactone) (PCL) (MW 43,000 to 50,000) in 10 mL of a mixed solution of chloroform and methanol (3: 1) The solution was injected into a syringe and connected to a syringe pump, and the distance between the syringe and the aluminum foil substrate was maintained at 15 cm The polycaprolactone solution from the syringe pump was electrospinned at a rate of 1 mL / The nanofibers were separated from the aluminum foil using 99% ethanol, and the separated nanofibers were milled for 30 seconds at a short end using a milling machine (IKA ^® A11 basic analytical mill) three times for 30 seconds. Was hydrolyzed with 40 mL of 1 molar M sodium hydroxide for 6 hours and rinsed three times with distillation to remove the salts. The length of the nanofibers was measured with an optical microscope And monitored every hour.

2. Preparation of hydrogels

Alginate and gelatin solution were respectively dissolved in a serum-free DMEM medium at a concentration of 4% and 5% (w / v), respectively. Then, alginate to gelatin was evenly mixed at a volume ratio of 2: 1 to prepare a mixed solution of alginate and gelatin Respectively. The polycaprolactone nanofibrils prepared in Section 1 above were sterilized by rotating in 70% ethanol overnight, and then rinsed three times with PBS. Then, polycaprolactone nanofibrils were mixed with alginate and gelatin (Alg-Gel) at a mixing ratio of 0, 1, 5, 10, 15 and 20% . Here, NIH3T3 cells, a kind of fibroblasts to be cultured, were added at a cell concentration of 10,000 cells per single hydrogel. Alginate, gelatin, nanofibrils, and cells were uniformly mixed, and the nanofibrils were crosslinked for 3 hours with a 0.5% calcium chloride solution to prepare a nanofiber-reinforced polymer hydrate gel complex.

EXPERIMENTAL EXAMPLE 1. Appearance of nano-fibril-reinforced polymer hydrogel complex

The appearance of the hydrogels containing nanofibrils at 0, 1, 5, 10, 15 and 20%, respectively, was observed. Figure 2 (a) shows a digital image of a nanofiber-reinforced polymer hydrate complex identified by a Canon EOS 450D DSLR camera. FIG. 2 (B) shows a scanning electron micrograph of a UHR-SEM (Ultra High Resolution Scanning Electron Microscope / Hitachi S-4800) instrument.

As shown in Fig. 2 (A), the color of the hydrogel became more clear white as the amount of nanofibrils increased, and it was confirmed that it became cylindrical.

As shown in FIG. 2 (b), it was confirmed that the density and degree of crosslinking of the nanofibrils increased with an increase in the amount of nanofibrils.

Experimental Example 2: Mechanical strength and weight loss of nano-fibril-reinforced polymer hydrate gel

1. Mechanical strength measurement

The mechanical strength of the nanofibril-reinforced polymer hydrate gel composite prepared in Example 1 containing nanofibrils at the ratios of 0, 1, 5, 10, 15 and 20%, respectively, was measured by a rotational rheometer (Gemini 200, Bohlin Instruments, Westborough, MA). Before and 14 days after culturing the cells

 The mechanical strength (Pa) after the incubation was measured and the results are shown in Table 1 below.

[Table 1]

Figure 3 (a) shows the elastic modulus before culturing the cells in the nanofibril-reinforced polymer-hydrated gel complex. Fig. 3 (B) shows the elastic modulus after culturing the cells for 14 days in the nanofiber-reinforced polymer-hydrated gel complex.

As shown in Fig. 3 (A), the increase of nanofibrils in the hydrogel when the cells were not added decreased the modulus of elasticity. From this, it was confirmed that the mechanical strength of the hydrogel was reduced as the amount of nanofibrils contained in the hydrogel increased before culturing the cells.

As shown in Fig. 3 (B), when the cells were added, the increase of the nanofibrils in the hydrogel increased the modulus of elasticity. After the cells were cultured, the mechanical strength of the hydrogel increased as the amount of nanofibrils contained in the hydrogel increased.

2. Measurement of weight loss rate

The weight loss rate of the nanofibril-reinforced polymer-hydrated gel composite prepared in Example 1 containing nanofibrils at the ratios of 0, 1, 5, 10, 15 and 20%, respectively, was measured using a METTLER TOLEDO AB204- And the results are shown in Table 2 below.

[Table 2]

Each of the nano-fortified hydrogels was lyophilized to measure the initial weight. The hydrogels containing NIH3T3 cells were cultured in DMEM containing 10% FBS for 14 days and then weighed to determine the weight loss rate (% ) Were obtained.

Weight Loss (%) = (Original Weight - Weight after Culture) / Original Weight X 100

3 (C) shows the result of measuring the weight loss rate (%) of the nanofiber-reinforced polymer-hydrated gel complex.

As shown in FIG. 3 (c), when the weight loss rate of the nanofibril-reinforced polymer-hydrated gel complex was measured, it was confirmed that the weight loss rate of the hydrogel decreased as the amount of nanofibrils increased.

Experimental Example 3: Cell survival rate in nanofiber-enhanced polymer hydrogel complex

Cell viability was measured by MTT assay to confirm the proliferation and growth of the cells in the nanofiber - reinforced polymer - hydrate complex.

The effect of the nanofibril-reinforced polymer hydrate complex on cell survival or growth was confirmed by cell culture in the nanofibril-reinforced polymer-hydrated gel complex prepared in Example 1. [

The nano-fibril-fortified polymer hydrogel complex, which contains nanofibrils at a ratio of 0, 1, 5, 10, 15 and 20%, respectively, and crosslinked with calcium chloride, was rinsed twice with PBS, and then FBS, penicillin and streptomycin (Culture medium). NIH3T3 cells were added to nano-fibril-fortified polymer-hydrate gel complex, and nano-fibril-enriched high-molecular-weight hydrogel complexes containing 1 × 10 ⁴ NIH3T3 cells were formed on 96-well plates. MTT solution (5 mg / ml) was added to 200 μl of medium containing no serum, followed by incubation for 4 hours in an incubator at 37 ° C. and 5% CO ₂ environment. After confirming the formation of formazan, 200 μl of DMSO was used to dissolve and the absorbance was measured at a wavelength of 540 nm. The viability of the cells was confirmed by MTT assay for 14 days while exchanging with fresh medium every 2 days, and NIH3T3 was detected at days 1, 3, and 14 Cell viability was measured. The cell viability was measured on the basis of the measurement result in a hydrogel not added with the first nano-fibril, and it is shown in Fig.

4 shows the survival rate (%) of the cells when the cells were cultured in the nano-fibril-reinforced polymer hydrogel complex for 14 days.

As shown in FIG. 4, it was confirmed that the cell viability was increased as the nanofibrils contained in the hydrogel were increased as compared with the hydrogel containing no nanofibrils.

EXPERIMENTAL EXAMPLE 4. Degree of Cell Stretch in Nanofibril-Reinforced Polymeric Hydrate Gel Complex

In order to confirm the extent of cell expansion in the nanofibril-reinforced polymer-hydrated gel complex prepared in Example 1 containing nanofibrils at the ratios of 0, 1, 5, 10, 15 and 20%, NIH3T3 Cells were incubated in an incubator at 37 < 0 > C, 5% CO ₂ for 7 days. Cell nuclei and actin were stained with 4,6-diamidino-2-phenylindole (DAPI) and phalloidin, respectively. After cells were fixed with 3.7% formaldehyde, 0.1% Triton X-100, a non-ionic surfactant, was used to permeabilize the cells with the dye.

 Cells and nanofibrils in the hydrogel were identified using a Multi-photon Confocal Laser Scanning Microscope (LSM 510META NLO / ZEISS) and cell images were obtained.

Fig. 5 (a) ((a) to (f)) shows the result of confocal laser microscopy of cells in a hydrogel containing nanofibrils at a ratio of 0 to 20%, respectively.

As shown in Fig. 5 (A), as the content of nanofibrils in the hydrogel increased, the extent of cell expansion increased. After confirming the appearance of the cells through a microscope, the degree of cell stretching was measured from the value obtained by dividing the long axis direction length of the cells in each image in Fig. 5 (A) by the short axis direction length as shown in the following formula.

(Extent of cell stretch) = (length of long axis of cell / length of short axis of cell)

FIG. 5 (B) shows the result of measuring the degree of cell stretching in the nanofibril-reinforced polymer hydrate gel complex.

As shown in FIG. 5 (b), it was confirmed that as the content of nanofibrils in the hydrogel increased, the value of the stretched degree obtained by dividing the long axis length of the cell by the short axis length increased.

In addition, the peripheral length of the cells was measured from each image in Fig. 5 (A). The peripheral length (탆) of the cells was obtained from the average value of the peripheral length of the cells by using Image J program, taking ten cells each present in the hydrogel containing nano-fibrils at a ratio of 0 to 20%. As shown in FIG. 5 (c), it was confirmed that as the nanofibril content in the hydrogel increased, the perimeter length of the cells increased.

Experimental Example 5. Gene Expression Confirmation in Nano-Fibril-Reinforced Polymeric Hydrogel Complex

The expression of collagen (type 1, 2) was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) in order to compare the expression level of collagen gene in the nanofiber-reinforced polymer hydrogel complex. Respectively.

After incubation, the cells were reacted for 5 min at 70 ° C for total RNA denaturation (total RNA denaturation) obtained from the hydrogel cells, and then 10X PCR buffer, Dntp mix, primer and Taq DNA polymerase (Taq.) Were used. The resulting cDNA was reacted at 37 ° C for 1 hour. 1.2% agarose gel was used for electrophoresis and loaded for 30 minutes at a voltage of 100v.

GAPDH and collagen type 1 and 2 gene primers as shown in Table 3 below were used.

[Table 3]

FIG. 6 shows the results of RT-PCR measurement of the gene expression of cells in the nano-fibril. ((A) to (f)): At the ratio of 0, 1, 5, 10, Fibril-containing hydrogels).

As shown in FIG. 6, it was confirmed that the expression of the collagen gene was relatively increased in the nanofibril-reinforced polymer hydrate gel complex containing 1 to 20% of fibrils as compared with the hydrogel having no nanofibrils (a).

<110> Industry-Academic Cooperation Foundation of Kangwon University <120> Nanofibril-reinforced polymer hydrogel complex and preparation          method thereof <130> PN118476 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 1 "s" <400> 1 ctccaacgag atcgagatc 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 1 "as" <400> 2 gtttackga agcagacagg 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 2 "s" <400> 3 gggtctcctg cctcctcctg ctc 23 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Collagen type 2 "as" <400> 4 tcctttctgc ccctttggcc ctaattttcg gg 32 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH "s" <400> 5 tcactgccac ccagaagac 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH "as" <400> 6 tgtaggccat gaggtccac 19

Claims (14)

Preparing nanofibers by electrospinning the hydrophobic polymer solution;
Hydrolyzing the nanofibers to produce nanofibrils;
Adding and mixing the nanofibrils to a hydrophilic polymer solution; And
And forming a cross-linking of the nanofibrils. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The hydrophobic polymer according to claim 1, wherein the hydrophobic polymer is selected from the group consisting of poly (ε-caprolactone) (PCL), poly (lactic acid): PLA, poly (glycolic acid) ), A polylactic-co-glycolic acid (PLGA), and a combination thereof. The method for producing a nanofiber-reinforced polymer-hydrated gel conjugate according to claim 1, The method of claim 1, wherein the hydrophilic polymer is selected from the group consisting of alginate, gelatin, hyaluronic acid, chitosan, pullulan, polyvinyl alcohol (PVA), poly (Γ-PGA), poly (γ-glutamic acid): γ-PGA, and combinations thereof. The method according to claim 1, wherein the hydrophilic polymer is a mixed solution of alginate (Alg) and gelatin (Gel). The method of claim 1, wherein the ratio of the mixed solution of alginate and gelatin is 3: 1 to 1: 3. The method according to claim 1, wherein the ratio of the mixed solution of alginate and gelatin is 2: 1. The method of claim 1, wherein the step of hydrolyzing the nanofibers is performed using sodium hydroxide (NaOH) solution or hydrochloric acid (HCl) as a catalyst. The method according to claim 1, wherein the step of forming the crosslinking of the nanofibrils comprises the step of forming a cross-linking group of calcium chloride (CaCl ₂ ), magnesium chloride (MgCl ₂ ), calcium phosphate (CaP), calcium carbonate (CaCO ₂ ) Is carried out with a cross-linking agent selected from the group consisting of polyvinylpyrrolidone and polyvinylpyrrolidone. [2] The method of claim 1, wherein the cross-linking step comprises a calcium chloride (CaCl ₂ ) solution as a cross-linking agent. [Claim 4] The method of claim 1, further comprising adding a target cell to the mixed solution between the step of adding the nanofibrils to the hydrophilic polymer solution and the step of forming the cross-linking of the nanofibrils, (I). [Claim 5] The method according to claim 1, further comprising the step of culturing the subject cells after the step of forming the crosslinking of the nanofibrils. [Claim 11] The method according to claim 10, wherein the target cell is a fibroblast. The nanofibril-reinforced polymer-hydrated gel composite produced by the method of claim 1. 14. The nano-fibril-reinforced polymer-hydrated gel complex according to claim 13,

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