KR101104305B1 - Fabrication Method of Polymeric Janus Microfiber having Porous Surface for Tissue Engineering - Google Patents

Abstract

The present invention relates to a method for manufacturing a polymer microfiber for tissue engineering having a porous surface and a manufacturing apparatus therefor. More specifically, a monomer jet stream is introduced into a microchannel in a continuous phase of an aqueous polymerizable photopolymerizable polyurethane monomer and an aqueous solution not mixed with the monomer. The present invention relates to a method for manufacturing a polymer microfiber, and a manufacturing apparatus for the same, wherein the photocured monomer is discharged in a continuous phase by irradiating ultraviolet rays to a microchannel while continuously being injected simultaneously.

According to the present invention, it is possible to manufacture polymer microfibers having a porous surface in real time by using a chemical reaction of monomers and water at the same time without photopolymerization and without a separate process for controlling the surface without using chemicals harmful to the human body. have.

Microfluidic Chip, Porous, Fiber, Support, Tissue Engineering

Description

Fabrication Method of Polymeric Janus Microfiber having Porous Surface for Tissue Engineering

The present invention relates to a method for producing a polymer microfiber for tissue engineering having a porous surface and a manufacturing apparatus therefor. More specifically, a method of manufacturing a polymer microfiber having a porous surface in real time by using a chemical reaction of monomer and water at the same time without photopolymerization and without a separate process for controlling the surface without using chemicals harmful to the human body. And it relates to a manufacturing apparatus for this.

In the United States, the nation's best health care nation, 100,000 patients need organs each year as part of their body, but only 20,000 organs are donated. About 36,000 people are waiting on the waiting list with conditions for organ transplantation, and about 10,000 have lost their lives in the last five years. Even though the human organs need a lot, the number of donors is gradually decreasing, causing severe imbalances in supply and demand, and many problems such as technical difficulties in organ transplant surgery, high cost, and side effects due to the use of immunosuppressants remain.

As a new approach to organ transplantation, the necessity for the development of artificial organs or tissue regeneration using tissue engineering has emerged. Tissue engineering integrates and applies the basic concepts and technologies of life sciences to understand the relationship between the structure and function of biological tissues, and to maintain, improve or restore the function of our bodies by creating and replacing the tissues. This is an applied research aimed at doing this. Representative tissue engineering techniques are summarized as follows. First, the necessary tissue is taken from the patient's body, the cells are separated from the tissue pieces, and the separated cells are grown in the required amount through culture. Proliferated cells are planted in a porous polymer scaffold and cultured in vitro for a period of time, and the hybrid cell / polymer structures obtained therefrom are transplanted again into the body.

The scaffold used in tissue engineering should not only have biocompatible properties because it does not cause transplant rejection, cytotoxicity, inflammatory reaction, etc., but also facilitates cell attachment and growth, and withstands in vivo pressure to serve as a scaffold. Should have high mechanical strength. Among scaffolds, fibrous scaffolds are widely used in tissue engineering because they can serve as guides in cell growth, alignment, and migration. Polyurethane as a support material in the form of fibers has the critical disadvantage that the adhesion of the cells is low despite the appropriate mechanical strength and biocpmpatibility. In order to make up for this drawback, a method of making a polyurethane support in the form of a fiber and then coating the surface of the support with a protein to which cells can adhere well is used, which is also difficult to uniformly coat the surface of the support. The process is complicated and has disadvantages in terms of time and cost.

Another important issue in tissue engineering is how to make a biocompatible scaffold. Conventional methods used to fabricate fibers include melt spinning and wet spinning, which use high temperature and volatile solvents to poison cells. There is a limitation as a method for preparing an engineering support.

Microfluidic chips, on the other hand, have fast reaction times, can be tested and analyzed with a small amount of reagents, and can integrate various processes in a small space. It is used. Polymer microfibers can also be manufactured in real time using microfluidic chips. Since high temperature and high pressure are not required, it is advantageous in terms of cost and space and can simplify additional devices. However, even in this case, it is difficult to control the surface properties of the microfibers in real time in the microfluidic chip. In order to use it as a support for tissue engineering, the microfibers are manufactured and then subjected to a separate post-treatment process. It should be made to be able to attach well.

In order to solve the above problems, an object of the present invention is to provide a method for producing a polymer microfiber for tissue engineering having a porous surface that is excellent in mechanical strength and easy to attach and grow cells.

It is another object of the present invention to provide a method for producing a polymer microfiber for tissue engineering having a porous surface economically by a simple process under mild conditions of room temperature and atmospheric pressure without using a volatile solvent that is toxic to cells.

Still another object of the present invention is to provide a manufacturing apparatus using a microfluidic chip capable of manufacturing a polymer microfiber for tissue engineering having a porous surface by the above method.

The present invention for solving the above problems and the photopolymerizable polyurethane monomer Simultaneously injecting an aqueous solution continuous phase not mixed with the monomer to form a monomer jet stream in a microchannel, while irradiating ultraviolet light to the microchannel to discharge the photocured monomer into the continuous phase. It relates to a manufacturing method.

In this case, when the monomer is first injected into the microchannel, it is more preferable to first fill the microchannel with the continuous aqueous solution before injection of the monomer in order to prevent the monomer from being adsorbed on the wall of the microchannel.

The photopolymerizable polyurethane monomer may be a mixture of a photoinitiator and a polyurethane monomer, and may be used by appropriately mixing a conventional photoinitiator and a polyurethane monomer known by the prior art, and may be used as a NOA63 (Norland optical adhesive 63) and NOA75 (Norland optical adhesive 75). Commercially available photopolymerizable polyurethane monomers such as the above may also be used.

The photoinitiator can form radicals by irradiation of ultraviolet rays, 1-Hydroxy-cyclohexyl-phenyl-ketone, 2-Hydroxy-2-methyl-1-phenyl-propan-1-one, 4- (2-hydroxyethoxy ) phenyl- (2-hydroxy-2-propyl) ketone, 2-Methyl-1 [4- (methylthio) phenyl] -2-morpholinopropan-1-one, 1-Hydroxy-cyclohexyl-phenyl-ketone, etc. It is not limited to this. The amount of photoinitiator may be appropriately adjusted according to the type of photoinitiator and the type of polyurethane monomer, but is preferably 1 to 5% (w / w) based on the polyurethane monomer.

The polyurethane monomer is a monomer compound or monomer composition capable of forming a polyurethane by a polymerization reaction, and may be any compound used as a general polyurethane monomer, and is usually composed of a mixture of polyisocyanate, glycol, and diamine. Polyurethane monomers generate urethane bonds, urea bonds, allophanate bonds, or biuret bonds by radicals generated from photoinitiators by ultraviolet irradiation, while hydroxyl groups are formed. When it comes into contact with the hydrophilic solution to contain, carbon dioxide gas is generated by reaction of the isocyanate group and the hydroxyl group of the polyisocyanate. The glycol is ethylene glycol, 1,2-propylene glycol, 1,3-butane diol, trimethylol propane, diamine is hydrazine, ethylene diamine, 1,4-ciclehexanediamine, 2,4-tolylenediamine, polyisocyanate p-phenylene diisocynate, 1,6-hexamethylene diisocyanate, toluene diisocynate may be exemplified, but this is only an example and the present invention does not apply only to specific polyurethane monomers. It may be easy to mix and use the polyurethane monomer. The mixing ratio of the glycol: diamine: polyisocyanate may be appropriately adjusted according to the prior art according to the physical properties of the polyurethane fiber to be finally obtained and the type of glycol, diamine and polyisocyanate to be used.

The continuous phase may be any aqueous solution that is not mixed with the monomer to form a monomer jet stream. That is, although water may be simply used, it is more preferable to use an aqueous solution of surfactant to lower the interfacial tension between the monomer and the continuous phase and to advantageously form a long monomer jet stream. The surfactant may be any one of ionic and nonionic, and those skilled in the art do not have any difficulty in selecting and using any of the conventional surfactants. It is preferable that the density | concentration of surfactant is 1 to 10% (w / w).

When a monomer and a continuous phase that are not mixed with each other meet, monomer droplets are formed at a location where the two fluids meet, and as the volumetric flow rate of the monomer increases, the monomer droplet is changed into a long cylindrical monomer jet stream. The volumetric flow rate varies depending on the width of the microfluidic channel, but the type of the continuous phase and the volumetric flow rate of the continuous phase. In addition, it will be easy for one skilled in the art to determine the volumetric flow rate of the monomer under which conditions the monomer forms a jet stream.

The isocyanate group of the polyurethane monomer at the interface of the continuous stream with the monomer jet stream formed in the microchannel reacts with water contained in the continuous phase to generate carbon dioxide (Scheme 1). The carbon dioxide gas thus generated floats upward in the opposite direction of gravity inside the monomer jet stream as shown in FIG. 3, and at the same time, the monomer jet stream is cured and discharged in real time. Therefore, some of the carbon dioxide gas is trapped inside the microfiber, and some of the carbon dioxide is hardened while being discharged from the upper surface, so that a small pore is formed on the surface. As a result, the upper surface is porous and the lower surface is non-porous. Microfibers are formed.

<Scheme 1>

R-NCO + H ₂ O → (RNHCOOH →) RNH ₂ + CO ₂ ↑

The present invention also relates to an apparatus capable of producing polymer microfibers by the above method. More specifically, the polymer microfiber 3 manufacturing apparatus 100 of the present invention includes a monomer injection port 1a into which a monomer is injected, a continuous phase injection port 1b into which a continuous phase is injected, as shown in FIG. A microfluidic chip comprising a junction point 2a for concentrating the continuous phase, a microfluidic channel 2 through which the focused monomer and the continuous phase flow, and an outlet port 2b for dispersing the focused monomer and the continuous phase; A water tank (5) in which the microfluidic chip is placed; And ultraviolet irradiation means 6 for irradiating ultraviolet rays to the microchannel of the microfluidic chip.

The width of the microfluidic channel in the microfluidic chip may be appropriately adjusted depending on the thickness of the microfibers to be manufactured and the type of the monomer, but is preferably 50 to 300 µm. If the width of the microchannel is too wide, a larger amount of continuous phase must be injected to maintain a uniform monomer jetstream in the microchannel. If the width is too narrow, the monomer jetstream comes into contact with the wall of the microchannel. There is a problem that the monomer jet stream cured by ultraviolet irradiation blocks the micro channel. In the present invention, since the irradiation of ultraviolet light is concentrated on a part of the micro channel, the length of the micro channel does not significantly affect the reaction.

The bath is filled with the photocured polymer microfibers and the continuous phase discharged from the microfluidic chip. At this time, it is preferable that the tank contains a continuous phase used in the manufacture of the microfibers in advance so that the conditions inside and outside the microfluidic chip do not change rapidly with the outlet 2b as a boundary.

As shown in FIG. 3, the monomer inlet 1a and the continuous phase inlet 1b may be respectively composed of two. The use of two continuous phase inlets positioned symmetrically with each other is more advantageous for forming a monomer jet stream because it can apply a uniform shear force to the left and right of the injected monomer flow. Meanwhile, when two kinds of monomers having different properties are injected into each injection hole through two monomer injection holes, microfibers having different properties on both sides may be manufactured. In the case of using one type of monomer as in the embodiment, having one and two monomer inlets has no significant effect on the results.

In addition, when the outlet of the microfluidic chip is directed in the vertical direction, the microfibers cured by ultraviolet rays may be blocked in the microfluidic channel, so that the outlet is directed in the horizontal direction to prevent this.

The invention also relates to a polymeric microfiber having a porous surface for tissue engineering prepared by the method. The polymer microfibers have a uniform thickness, and as shown in FIGS. 4 to 6, half of the surface has a porous structure and the other half has a non-porous Janus structure. The porous structure, which is half of the microfibers, facilitates cell attachment and growth, and the other half of the surface having the non-porous structure maintains the mechanical strength of the microfibers more strongly so that the microfibers prepared by the present invention It is intended to exhibit more suitable properties as an engineering support.

As a result of culturing NH3T3 cells using the polymer microfiber of the present invention as a cell culture support, the cells began to adhere to the porous surface of the microfiber at first, and then non-porous surface branched cells were attached after 36 hours. It was confirmed that the cells grew. In addition, the polymer microfibers are intersected and grown in a thin membrane form, and as the cultivation time passes, the membrane distance is gradually widened, and it can be applied to not only 2D cell culture but also 3D cell culture and research. .

According to the production method and apparatus of the present invention as described above, it is possible to continuously produce a polymer microfiber having a porous surface of uniform thickness by a simple process without using a volatile solvent that is toxic to cells at room temperature and atmospheric pressure. have.

In addition, the polymer microfiber having a porous surface according to the present invention maintains the excellent mechanical strength of the polyurethane and is easy to attach and grow cells due to the porous surface structure, and as a cell culture support in tissue engineering without a separate surface treatment Can be used.

In addition, since the cells are stretched and grown in the form of a thin film at the portion where the polymer microfibers intersect, the present invention can be applied to cell culture and research of 3D cells as well as 2D cells.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings and examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

Example 1 Preparation of Microfiber Manufacturing Apparatus

The microfluidic equipment is designed to have a microchannel having two inlets 1a and 1b and one outlet 2b as shown in FIG. The negative photosensitive agent (SU-8, Microchem Co., USA) was evenly applied on the silicon wafer, and then spin-coated at 1,000 rpm to raise the 100 μm high photosensitive agent. Width The UV mold was irradiated through a mask having a micro flow path having a thickness of 200 μm to prepare a master mold having a shape opposite to that of the channel. Then, PDMS (Polydimethylsiloxane) (Sylgard 184; Dow Corning, Midland, MI) was poured into the prepared master mold and then 4 hours at 65 ℃ Curing to produce a PDMS mold having a microchannel of the desired shape. The glass substrate was attached to the PDMS mold thus prepared through an oxygen plasma treatment to produce a microfluidic chip 100 for microfiber production. Tygon tube is inserted into the two inlets through a hole, and each tube is connected with a micro syringe (Norm-Ject, Germany), and the syringe pump (Harvard Apparatus, USA) is used to finely dispense the solution. Allow pumping into the flow path. The microfluidic chip 100 was placed in a tank 5 filled with a continuous phase 4 prepared in advance . In order to use an ultraviolet lamp connected to a fluorescence microscope as an ultraviolet irradiation device, the tank is placed on a microscope stage (NIKON, T2000-U, Japan), and ultraviolet rays are generated at a portion where a monomer jet stream is formed by the flow of a continuous phase and a monomer. Positioned to be irradiated to complete the micro fiber manufacturing apparatus.

Example 2 Preparation of Polymer Microfibers with Porous Surfaces

Using a microfluidic device prepared in Example 1 while irradiating ultraviolet rays with a 365 nm ultraviolet lamp (Osram, Germany) mounted on a microscope, one of the inlets (1a), using a syringe pump 5% SDS solution Injection was made at a rate of 10 μl / min. When the microchannel 2 was completely filled with SDS aqueous solution, NOA63 (Norland optical adhesive 63) was simultaneously injected into the other inlet (1b) using a separate syringe pump while continuously injecting the SDS aqueous solution. . NOA63 is an adhesive whose monomer is a precursor of polyurethane and is solidified by ultraviolet rays.

FIG. 4 is a microscope (NIKON, T2000-U, Japan) photograph of the polyurethane microfibers prepared by the above method, in which microfibers having a uniform thickness of about 50 μm in diameter are not produced without inter-fiber adhesion. I could confirm it.

The surface structure and the internal cross section of the microfibers manufactured by Scanning Electron Microscope (TOPCON SM-500, Japan) were observed and the photographs are shown in FIGS. 5 and 6, respectively. As can be seen in Figure 5, the surface structure and the internal cross-sectional structure of the polyurethane microfiber prepared by the present invention was confirmed through the photograph of the manufactured fiber (Figs. 5 and 6). As a result, it was confirmed that half of the cross section perpendicular to the longitudinal direction of the fiber produced by the present invention is a microfiber having a porous structure and the other half having a smooth surface without holes.

Example 3 Cell Culture with Microfiber Support

The polyurethane microfibers prepared in Example 2 were cultured with NIH3T3 cells as a support to confirm that they had suitable properties as a support for tissue engineering.

First, the microfibers prepared in Example 2 were washed with 70% ethanol, sterilized and dried. The microfibers were placed in a cell culture dish, and the medium solution in which the NIH3T3 cells were dispersed was poured thereon so that the cells adhered to the fiber surface. Here, DMEM (Dulbecco's Modified Eagle Medium) containing fetal bovine serum (FBS), penicillin, and streptomycin was used. Cells were cultured in a cell culture incubator (Sanyo CO ₂ incubator MCD-18AIC, Japan) maintained at 5% carbon dioxide and 37 ℃ temperature conditions.

The growth of cells attached to the microfibers and the survival of the cells during the culture period were observed by optical and fluorescence microscopy (NIKON, T2000-U, Japan) and scanning electron microscope (TOPCON SM-500, Japan). 7 is shown. In addition, the ratio of the adhesion area of the cells according to the incubation time was calculated by dividing the porous region and the non-porous region of the microfiber and the results are shown in the graph of FIG. 8. As can be seen in Figures 7 and 8, in the early stage of cell culture, the cells adhere to the porous surface of the microfibers and grow, and after 36 hours of incubation, the cells move to the surface without the porous structure, and the growth occurs. Cells attached to and grew on most surfaces. 9 is a graph showing the survival rate of the cells calculated by Live / Dead Assay of the cells using a fluorescence microscope according to the culture time, and by confirming that the living cells fluoresce green, most of 90% or more after 120 hours It was confirmed that the cells were alive.

As a result of prolonged incubation period and growing by optical microscope, it was confirmed that the cells grew well on the support for 15 days of cultivation. (FIG. 9). 10 is a graph measuring the distance of the membrane formed between the polymer microfibers according to the culture period, it was confirmed that the distance of the membrane is also gradually increased during the culture period of 15 days. Through this, it was confirmed that the polymer microfiber support made according to the present invention can be applied to cell culture and research of 3D type as well as 2D type cell culture.

1 is a schematic diagram and a reaction scheme showing the formation mechanism of the polymer microfiber having a porous surface according to an embodiment of the present invention.

2 is a cross-sectional view of a micro fiber manufacturing apparatus according to an embodiment of the present invention.

Figure 3 is a perspective view of a micro fiber manufacturing apparatus according to another embodiment of the present invention.

Figure 4 is an optical micrograph of the microfiber prepared according to the embodiment of the present invention.

5 is a scanning electron micrograph showing the surface structure of the microfiber prepared according to the embodiment of the present invention.

6 is a scanning electron micrograph showing the cross-sectional structure of a microfiber prepared according to an embodiment of the present invention.

Figure 7 is a fluorescence microscope, optical microscope, scanning electron micrographs according to the culture time when the cells were cultured using the microfiber prepared according to the embodiment of the present invention as a support for cell culture.

Figure 8 is a graph showing the area ratio of the cells on the surface of the porous structure and the surface of the non-porous structure for each culture time when the cells were cultured using the microfiber prepared according to the embodiment of the present invention as a support for cell culture .

Figure 9 is a graph showing the survival rate of cells for each culture time when the cells were cultured using the microfibers prepared according to the embodiment of the present invention as a support for cell culture.

10 is an optical micrograph of each culture time when the cells are cultured for a long time using the microfibers prepared according to the embodiment of the present invention as a support for cell culture.

Figure 11 is a graph showing the maximum distance of the membrane formed by the growth of the cells by the culture time when the cells are cultured when the cells are cultured using the microfibers prepared according to the embodiment of the present invention as a cell culture support.

** Description of the symbols for the main parts of the drawings **

100: micro fiber manufacturing apparatus

1a: monomer injection port 1b: continuous phase injection port

2: fine flow path

   2a: junction point of continuous phase and monomer 2b: outlet

3: polymer microfiber

4: Continuous phase

5: tank

6: ultraviolet irradiation means

Claims (11)

While simultaneously injecting a photopolymerizable polyurethane monomer and an aqueous solution continuous phase which is not mixed with the monomer to form a monomer jet stream in a microchannel, Irradiating the ultraviolet light to the micro-channel so that the photocured monomer is discharged in a continuous phase, A cross-sectional reference half of the microfiber surface is a porous structure, and the other half has a non-porous structure. The method of claim 1, The method of manufacturing a polyurethane microfiber for tissue engineering, characterized in that to fill the micro-channel first with the continuous aqueous phase solution before the injection of the monomer. The method according to claim 1 or 2, The photopolymerizable polyurethane monomer is a method for producing a polyurethane microfiber for tissue engineering, characterized in that the mixture of the photoinitiator and the polyurethane monomer. The method according to claim 1 or 2, The photopolymerizable polyurethane monomer is NOA63 (Norland optical adhesive 63) characterized in that the manufacturing method of polyurethane microfibers for tissue engineering. The method according to claim 1 or 2, The continuous phase is a method for producing a polyurethane microfiber for tissue engineering, characterized in that the aqueous surfactant solution. delete delete delete delete delete A cross-sectional reference half of the microfiber surface is a porous structure, and the other half has a nonporous structure. Polyurethane microfiber for tissue engineering.

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