KR101667406B1 - Hydrophilic nanofiber scaffold comprising PVP-b-PCL block copolymers - Google Patents

Abstract

The present invention relates to a hydrophilic nanofiber cell support comprising a PVP- b- PCL block copolymer and a process for its preparation. Since the hydrophilic nanofiber cell scaffold containing the PVP- b- PCL block copolymer according to the present invention has no cytotoxicity and has an excellent effect of increasing cell adhesion and proliferation by surface hydrophilization, Can be used for the development of extracellular matrix (ECM) biomaterials derived from skin cells through reconstitution or degassing, and can be usefully used as a cell scaffold for tissue engineering in various fields.

Description

Hydrophilic nanofiber scaffolds comprising PVP-b-PCL block copolymers are disclosed.

The present invention relates to a hydrophilic nanofiber cell support comprising a PVP- b- PCL block copolymer and a process for its preparation.

Biotissue engineering is a series of techniques that induce normal function by restoring, regenerating, and replacing new parenchyma by transplanting a biocompatible polymer scaffold and cells that are completely absorbed in the living body and have cell affinity. This is a multi-disciplinary combination of cell cultivation, material engineering, and transplant surgery. Using these tissue engineering techniques, desired tissue can be obtained and the shape can be molded at will according to the shape of the support. In the field of tissue engineering, biological signals of cells and scaffolds are very important factors, and the role of biomaterial scaffolds in use should be to help cells to settle and culture well in vivo, Should be distributed uniformly on the support, and the separated cells should promote the regeneration of the differentiated tissue by abundantly releasing extracellular matrix (ECM) in vivo. In addition, after transplantation into the body, it should be biocompatible to absorb and decompose safely after a certain period of time.

The ideal support for effective tissue regeneration is the requirement of sufficient mechanical stress to withstand body loading, the ability to adhere and grow cells, the micro and macro structure of pores, size and shape. The scaffold plays an important role in the growth of cells that migrate from seeded cells and tissues. Most cells in the body are adherent cells. If there is no place to attach, the cells do not grow and die. Due to the characteristics of these cells, there are many difficulties in the treatment of cells. To solve this problem, supports have been introduced and widely used.

The polymers used in the above-mentioned tissue engineering supports can be roughly divided into natural polymers and synthetic polymers, and they have suitable physico-chemical, biological and mechanical properties. Natural polymers include collagen, hyaluronic acid, alginate, gelatin, xanthan gum, keratin, and small intestinal submucosa, which have excellent biocompatibility and low immune responses after transplantation. However, natural polymers have disadvantages in that they do not have sufficient mechanical properties when used individually.

The synthetic polymers are mainly hydrophobic polyesters such as poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (lactic-co- glycolic acid) (PLGA) and poly (e-caprolactone) Of these, PGA, PLA, and PLGA, which are α-hydroxy acid-based polymers, are synthesized polymers approved by US FDA and widely used as biomaterials such as tissue-engineered porous supports and drug delivery systems, and have high biocompatibility and processability Lt; / RTI > However, it is difficult to attach cells due to the lack of bioactive substances and hydrophobicity, and the acid decomposition products generated during the hydrolysis process have a drawback of decreasing the pH around the tissues and causing inflammation.

On the other hand, nanofiber supports of biodegradable polymers such as PCL, which are mainly used as cell supports, have a limitation of initial cell adhesion due to the hydrophobicity of the polymer itself.

The present inventors have conducted studies to develop new nanofiber cell scaffolds that solve the above problems. As a result, it has been found that nanofiber cell scaffolds containing PVP- b- PCL block copolymer are hydrophilized on the surface, And proliferation, thereby completing the present invention.

It is an object of the present invention to provide a hydrophilic nanofiber cell support comprising a PVP- b- PCL block copolymer and a process for its preparation.

In order to achieve the above object, the present invention provides a hydrophilic nanofiber cell scaffold comprising a PVP- b- PCL block copolymer and a method for producing the same.

Since the hydrophilic nanofiber cell scaffold containing the PVP- b- PCL block copolymer according to the present invention has no cytotoxicity and has an excellent effect of increasing cell adhesion and proliferation by surface hydrophilization, Can be used for the development of extracellular matrix (ECM) biomaterials derived from skin cells through reconstitution or degassing, and can be usefully used as a cell scaffold for tissue engineering in various fields.

1 is a block diagram showing a result of observation through a PCL / PVP -b- PCL nanofibers SEM of the invention [PCL / PVP-b-PCL (w / w): (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, (e) 70/30].
2 is a graph showing the contact angle according to the content of the PCL / PVP- b- PCL nanofiber of the present invention.
FIG. 3 is a graph showing changes in the surface of the PCL / PVP- b- PCL nanofibers of the present invention after the dissolution experiment.
4 is a graph showing the effect of PCL / PVP- b- PCL nanofibers of the present invention on cell adhesion.
FIG. 5 is a graph showing the attachment pattern of cells in the PCL / PVP- b- PCL nanofiber treatment of the present invention. FIG.
6 is a graph showing the results of measurement of cytotoxicity of the PCL / PVP- b- PCL nanofibers of the present invention.
7 is a graph showing the effect of PCL / PVP- b- PCL nanofibers of the present invention on cell activity.
FIG. 8 is a graph showing the effect of the PCL / PVP- b- PCL nanofibers of the present invention on cell distribution through fluorescence staining.

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

The present invention provides a hydrophilic nanofiber cell support comprising a PVP- b- PCL block copolymer.

The cell support comprises a biodegradable polymer together with a PVP- b- PCL block copolymer. The polymer may be at least one selected from the group consisting of PCL, PLA, PGA, PLGA, diol / diacid aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxybutyrate) ), Polyanhydrides, polyorthoesters, polyurethanes, poly- N -isopropylacrylamides and derivatives or copolymers thereof, preferably PCL.

The biodegradable polymer and the PVP- b- PCL block copolymer may be mixed at a weight ratio of 99: 1 to 70:30, preferably 85: 95: 5-15. By mixing in the above ratio, it can exhibit the most suitable hydrophilicity for cell culture at the time of production.

The present invention also provides a method for producing a nanofiber mat comprising the steps of (1) dissolving a PVP- b- PCL block copolymer and a biodegradable polymer in an organic solvent, and (2) ; And a PVP- b- PCL block copolymer.

More specifically, a biodegradable polymer, preferably PCL, is dissolved in an organic solvent and a certain amount of PVP- b- PCL block copolymer is added. After sufficiently mixing the solution, the nanofiber mat is fabricated using an electrospinning apparatus. At this time, the needle is preferably 15 to 20 gauge, the distance from the collector to the collector is preferably 10 to 20 cm, and the electrospinning voltage intensity is preferably 10 to 20 kV.

The organic solvent includes tetrahydrofuran, dimethylformamide, dichloromethane, chloroform, acetone, dioxane, trifluoroethane, hexafluoroisopropane, a mixed solvent thereof, and the like.

The cell scaffold containing the PVP- b- PCL block copolymer of the present invention is free from cytotoxicity and has an excellent effect of increasing cell proliferation under cell attachment and high concentration culture conditions by surface hydrophilization. In addition, since the hydrophilic PCL cell scaffold comprising the PVP- b- PCL block copolymer of the present invention contributes to the stable growth of cells and the formation of tissues in the culture environment constituted by the cell scaffold, And can be used as a complex ECM. Therefore, the cell scaffold comprising the PVP- b- PCL block copolymer of the present invention can be used as a cell scaffold for tissue engineering.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and experimental examples.

Example  1. PVP -b- Preparation of Cellular Supports Containing PCL Block Copolymers

1-1. PVP -b- Synthesis of PCL block copolymer

[CL] ₀ / [VP] ₀ / [HECP] ₀ / [DPP] ₀ / [V-70] ₀ = 200/200/1/2 / 0.4: ε-caprolactone (DPP, 0.0688 g, 27 mmol) was dissolved in 3 mL of anisole at room temperature, and the mixture was stirred at room temperature for 2 hours. (anisole) and the mixture was stirred at 30 < 0 > C for 6 hours. Then, 2,2'-azobis (4-methoxy-2,4-dimethylvaleronitrile) (V70, 0.0170 g, 0.055 mmol) N -vinylpyrrolidone (VP, 2.94 mL, 27 mmol) dissolved in N, N- dimethylformamide (50 mL) was added to the reaction mixture and stirred for 12 hours at 30 ° C. The reaction was then submerged in diethyl ether It was then dried in a vacuum oven for 24 hours to recover the copolymer PVP- b -PCL block of polymerized PVP- b -PCL molecular weight of the block copolymer Mn = 26,300 g / mol, a molecular weight dispersity was 1.04 (PDI) The ratio of PVP to PCL in the PVP- b- PCL block copolymer measured by nuclear magnetic resonance spectroscopy ( ¹ H-NMR) was found to be 1: 1.35 by gel permeation chromatography (GPC) Respectively.

1-2. Electric radiation

PCL (Mw = 80,000 g / mol, Sigma Aldrich) and the PVP- b- PCL block copolymer prepared in Example 1-1 were weighed at 100/0, 90/10, 85/15, 80/20 , And 70/30 (w / w), respectively, to prepare spinning solutions having concentrations of 11.0%, 11.5%, 12.3%, 13.0% and 15.0% Nanofibers were prepared. A THF / DMF (1/1 v / v) mixed solvent was used as the electrospinning solvent. The voltage at the time of electrospinning was 13 kV, the distance between the collector and the nozzle was 13 cm, and the flow rate was 0.8 ml / h. The viscosity of each solution was measured at this time, and it is shown in Table 1.

PCL / PVP- b- PCL (w / w) Concentration of solution (%) Viscosity (cp) 100/0 11.0 161 90/10 11.5 148 85/15 12.3 141 80/20 13.0 140 70/30 15.0 142

Experimental Example 1. Characterization of the prepared cell scaffold

1-1. SEM analysis and average fiber diameter measurement

A scanning electron microscope (SEM) (Hitachi S-4300) was used to observe the shape of the nanofibers prepared in Example 1. The results are shown in FIG.

As shown in FIG. 1, it was confirmed that nanofibers were well formed, and the average fiber diameter was determined by measuring the diameters of twenty fibers, which are shown in Table 2.

PCL / PVP- b- PCL (w / w) Average diameter (nm) 100/0 476.74 + - 31.70 90/10 407.78 ± 20.36 85/15 422.33 + 26.81 80/20 421.34 ± 28.99 70/30 438.25 + - 24.56

As shown in Table 2, it was confirmed that the average fiber diameters of the nanofibers were 476.74, 407.78, 422.33, 421.34 and 438.25 nm, respectively, which are similar to each other.

1-2. Contact angle measurement

The contact angle was measured in order to measure the degree of hydrophilization according to the content of PVP- b- PCL of the nanofiber prepared in Example 1, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that as the content of PVP- b- PCL increased, the contact angle decreased and the hydrophilicity of the nanofiber mat was increased. As the affinity for water increased, Was also accelerated.

1-3. Dissolution experiment

It was experimentally examined whether PVP, a hydrophilic material, was eluted from the culture environment of the cell. PCL / PVP- b- PCL (70/30 w / w) nanofiber cell scaffolds were placed in an oven at 30 ° C for 1 hour to 10 days, respectively, immersed in water. Then, the nanofiber cell supporter was washed with methanol and vacuum dried to compare the weight before and after the experiment. The results are shown in Table 3.

In addition, SEM analysis was performed to observe whether the shape of the surface of the nanofibers was changed after the elution test, and the result is shown in FIG.

Weight (mg) I'm after 1 hours 6.6 6.1 3 hours 6.4 6.2 6 hours 6.2 6.0 12 hours 6.3 6.0 1 day 6.7 6.7 3 days 6.6 6.6 5 days 6.8 6.2 7 days 6.6 6.4 10 days 6.7 6.4

As shown in Table 3 and Fig. 3, there was no suspected weight change due to the elution of the hydrophilic material, and the nanofiber shape was also maintained. Therefore, it was confirmed that PCL / PVP- b- PCL nanofiber cell scaffold maintains initial hydrophilicity even in cell culture.

Experimental Example 2. Observation of cell support

SEM was used to observe the shape of the nanofiber cell supporter prepared in Example 1 attached to the cells. Cells were used in primary cultures of SD rats. Primary cultures were prepared by cutting the skin of 1-day old SD rats (1 cm * 1 cm), placing them in HBSS medium containing 1 mg / ml of Dipassa, reacting at 4 ° C for 24 hours, And the epidermal layer were separated. This was added again to HBSS medium containing collagenase 2 (1 mg / ml) and reacted at 4 ° C for 24 hours. Each epidermis and dermis were filtered through a cell filter with a pore size of 70 μm and cultured in high glucose-DMEM medium (10% FBS, 1% antibiotic). The PCL / PVP- b- PCL nanofiber mat or PCL nanofiber mat prepared in Example 1 was placed on a 24-well plate, seeded at a concentration of 1 × 10 ⁴ cells / well, and cultured for 5 days. After 5 days, each nanofiber mat sample was washed with PBS, and 3% glutaraldehyde was added, followed by incubation at 37 DEG C for 30 minutes. Thereafter, the concentration of ethanol was increased to 25%, 50%, 75%, 90%, and 100% and dehydrated. Three drops of hexamethyldisilazane (HMDS) were dropped on each of the dewatered nanofiber mat samples, followed by reaction for 3 minutes, followed by evaporation in a vacuum for 30 minutes. The results are shown in FIG. 4 Respectively.

As shown in Fig. 4, it was confirmed that the cells were more stably attached to the nanofiber mat of the present invention as compared with the PCL nanofiber mat.

On the fourth day of incubation, the cell attachment pattern in each sample was observed at a magnification of 1 to 10 K, and the results are shown in FIG.

As shown in Fig. 5, it was confirmed that cells adhered to the support as nanofibers in all the samples.

Experimental Example  3. Of cell support  Cytotoxicity analysis

(MTT assay, 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyltetrazolium-bromide, Sigma-Aldrich, St. Louis, MO) for the cytotoxicity of the nanofiber cell scaffold prepared in Example 1. Louis, MO). More specifically, the PCL / PVP- b- PCL nanofiber mat or the PCL nanofiber mat prepared in Example 1 was placed in a 24-well plate and DMEM medium (1% antibiotic) was added and cultured for 1 day. Cells were seeded at a concentration of 3x10 ³ cells / well in a 96-well plate, followed by addition of a medium prepared by previously preparing each nanofiber sample and culturing for 1 day. Thereafter, each well was treated with MTT solution, dissolved in DMSO, and then absorbance was measured at OD 540 nm. The results are shown in Fig.

As shown in Fig. 6, the medium treated with the PCL / PVP- b- PCL nanofiber mat of the present invention showed similar values to the medium treated with the PCL nanofiber mat which was already known to be non-toxic. Respectively.

Experimental Example  4. Cell activity measurement

The cell activity of the nanofiber cell scaffold prepared in Example 1 was assayed by MTT assay. Cells were seeded at a density of ³ × 10 ³ cells / well in a 96-well plate and cultured for 1 day. Each well was replaced with a DMEM medium containing the PCL / PVP- b- PCL nanofiber cell supporter prepared in Example 1 (0 to 100 μg / mg), and 1, 3 and 5 days later, MTT The activity of the cells was measured by the assay, and the results are shown in FIG.

As shown in Fig. 7, inhibition of the decrease in cell activity was confirmed in cells treated with the cell supporter, and a cell support having a ratio of PCL and PVP- b- PCL block copolymer of 9: 1 was most suitable for cell culture Respectively.

In addition, the cell distribution of the 9: 1 sample exhibiting the best effect was confirmed at intervals of 1 day, 4 days, and 7 days through fluorescent nuclear staining, and the results are shown in FIG.

As shown in FIG. 8, it was confirmed that the density of the cells was increased with an increase in time. In particular, on day 7, aggregates of cells were formed.

Through the above experiment results, it was confirmed that the cells well adhered proliferation on PCL / PVP -b- PCL nanofiber of the present invention, by using the PCL / PVP -b- PCL nanofibers into cells support the formation of the artificial tissue And it is confirmed that it is possible to produce a complex ECM that can be used as an insert in a human body when removing cells.

Claims (9)

A nanofiber cell scaffold comprising PCL and poly (N-vinylpyrrolidone) -b-poly (ε-caprolactone) block copolymer as a 9: 1 (w: w) block copolymer. delete delete delete The nanofiber cell scaffold of claim 1, wherein the cell support is hydrophilic. (a) dissolving PCL and PVP-b-PCL block copolymer in an organic solvent at a ratio of 9: 1 (w: w); And
(w: w) containing PCL and PVP-b-PCL block copolymers, wherein the step (b) comprises electrospinning the mixture of step (a) A method for producing a fibrous cell support. delete The method of claim 6, wherein the organic solvent in step (a) is selected from the group consisting of tetrahydrofuran, dimethylformamide, dichloromethane, chloroform, acetone, dioxane, trifluoroethane, hexafluoroisopropane, Wherein the hydrophobic nanofibrous cell support is at least one selected from the group consisting of nanoparticles and nanoparticles. 7. The method of claim 6, wherein the electrospinning in step (b) is performed under a voltage of 10-20 kV.

Images