KR101333381B1 - Preparation method of bilayer scaffold in one-step and tissue regeneration method of bilayer scaffold obtained thereby - Google Patents

Abstract

The present invention relates to a method for producing a double layer scaffold by a single process and to a tissue regeneration method using a double layer scaffold obtained by the method, wherein the method for preparing a double layer scaffold is prepared by attaching each polymer scaffold separately. Unlike the conventional method of manufacturing a double layer scaffold manufactured in a two-step process through the process to be able to manufacture a double layer scaffold of the completely attached form through a single process without a separate attachment process. In addition, the cells are cultured in the bilayer scaffold thus prepared and applied to tissue regeneration of the skin defect area, or cocultured with chondrocytes and bone cells to areas where tissues are in contact with each other, such as bone and cartilage, to promote tissue regeneration. Can be.

Description

Preparation method of bilayer scaffold in one-step and tissue regeneration method of bilayer scaffold obtained}

The present invention relates to a method for producing a double layer scaffold by a single process and to a tissue regeneration method using the double layer scaffold obtained by the method.

Scaffolds refer to physical scaffolds and adhesive substrates made for in vitro culture and transplantation of tissue cells. These scaffolds are used for cell transplantation for human tissue regeneration, and also for mass culture and proliferation of cells. The importance is very high. This is due to the adhesion of the cells at the contact area between the cells and the substrate and the subsequent migration and proliferation of epithelial cells.

In other words, most biologically active cells have a basic step that must be passed in order to survive in contact with substances in or outside the body. The first step is cell adhesion. In particular, when looking at the survival stage of fibroblasts and tissue cells, the cells preferentially adhere to the substrate, and after adhesion, the metabolism of organelles in the cytoplasm becomes active, and a new site is used to facilitate the proliferation and supply of nutrients. Will move.

Thus, the surface that activates the deposition of cells is the most basic means of doubling cell proliferation. The adhesion of these cells to the substrate can be artificially controlled by the components of the substrate. Scaffolds are carriers that support scaffolds as they regenerate and grow, that is, the basis of artificial substrates. Recently, scaffolds have been used in mass culture and growth vessels or flasks of cells.

On the other hand, Korean Patent Laid-Open No. 2004-0016984 relates to a carrier for culturing cells and tissues and a method for culturing, wherein the carrier for culturing cells and tissues that can effectively regenerate a target tissue or organ while suppressing overproliferation of fibroblasts, And cell and tissue culture methods. However, the scaffolds known in the patent are not only excellent in adhesion and physical properties to the substrate to be suitable for mass culture of the cells, but also have many problems in that the manufacturing process is not simple and undesirable.

In addition, in manufacturing a conventional double layer scaffold, a double layer scaffold was manufactured by attaching each polymer scaffold separately and then attaching the double layer scaffold. There is a problem that the process is complicated because it goes through a two-step process of making a thick and individual polymer scaffold.

Accordingly, the present inventors completed the present invention by confirming that the scaffold obtained by adding a surfactant to a solution in which two polymers are blended to cause phase separation through a high temperature and high speed stirring reaction is a structurally clear double layer scaffold.

Accordingly, it is an object of the present invention to provide a method for producing a bilayer scaffold that is not separated through a simple single process.

Another object of the present invention is to provide a tissue regeneration method using the bilayer scaffold.

In order to achieve the above object, the present invention comprises the steps of preparing a first polymer aqueous solution; Adding and stirring a second polymer to the first aqueous polymer solution; Adding a surfactant to the stirred reactant to perform high temperature and high speed stirring; Lyophilizing the stirred reactant to obtain a sponge; Dipping the sponge in a crosslinking agent to crosslink it; And it provides a method for producing a double-layer scaffold by a single process comprising the step of lyophilizing the crosslinked reactant again.

The first polymer and the second polymer are different polymers , gelatin, Chitosan, elastin, hyaluronic acid, hydroxyapatite, alginate, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), poly [(Lactic-co- (glycolic acid)) (PLGA), poly [(3-hydroxybutyrate) -co- (3-hydroxyvalorate) (PHBV), polydioxanone (PDO), poly [(L Lactide) -co- (caprolactone)], poly (esterurethane) (PEUU), poly [(L-lactide) -co- (D-lactide)], poly [ethylene-co- (vinyl alcohol )] (PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS) and polyaniline (PAN). Preferably, the first polymer may be gelatin, and the second polymer may be polyvinyl alcohol (PVA).

The surfactant is sodium dodecyl sulfate (sds), ammonium lauryl sulfate (als), sodium lauryl ethylene sulfate (sles), linear alkylbenzene sulfonate (linear alkylbenzene sulfonate) Sulfonate (LAS), α-Olefin Sulfonate (AOS), Alkyl Sulfate (AS), Alkyl Ether Sulfate (AES) and Sodium Alkane Sulfonate (SAS) It may be selected from the group consisting of, and may be preferably sodium dodecyl sulfate (SDS).

The high temperature and high speed stirring is preferably stirred at 800 to 2000 rpm at a temperature of 80 to 120 ℃.

The crosslinking agent may be selected from the group consisting of glutaraldehyde, ethyldiethylaminopropylcarbodiimide (EDC), genepin, and transglutaminase (TG).

In addition, the present invention comprises the steps of preparing a gelatin aqueous solution by stirring at 100 to 300 rpm at 60 to 70 ℃ so that the gelatin is completely dissolved in water; Adding and stirring polyvinyl alcohol (PVA) to the gelatin aqueous solution; Adding sodium dodecyl sulfate to the stirred reactant at a high temperature and high speed at 80 to 120 ° C. to 800 to 2000 rpm; Lyophilizing the stirred reactant to obtain a sponge; Dipping the sponge in a crosslinking agent selected from glutaraldehyde or EDC (Ethyldiethylaminopropylcarbodiimide) to crosslink the sponge; And lyophilizing the crosslinked reactant again.

The present invention provides a tissue regeneration method comprising culturing cells in a bilayer scaffold according to the preparation method.

The cells may be any one or two or more selected from the group consisting of chondrocytes, bone cells and skin cells, and the cells may be co-cultured by culturing different cells in the upper and lower layers of the bilayer scaffold, respectively. have.

According to the manufacturing method of the present invention, unlike the conventional method of manufacturing a double-layer scaffold manufactured in a two-step process by manufacturing and attaching each polymer scaffold separately, it is completely attached through a single process without a separate attachment process The double-layer scaffold of the present invention can be prepared, and the cells are cultured in the double-layer scaffold thus prepared, and applied to tissue regeneration of the skin defect area, or the co-culture of chondrocytes and bone cells is brought into contact with tissues such as bone and cartilage. It can be applied to the part where it exists to promote tissue regeneration.

1 is a flowchart illustrating a manufacturing process of a double layer scaffold according to the present invention;
2 is a digital image of a bilayer scaffold in accordance with the present invention,
3 is a scanning electron microscope image of a bilayer scaffold according to the present invention,
Figure 4 is a scanning electron micrograph of cells cultured for one day in a bilayer scaffold according to the present invention,
5 is a scanning electron micrograph of cells cultured for 5 days in a bilayer scaffold according to the present invention.

Hereinafter, gelatin is used as the first polymer, polyvinyl alcohol (PVA) is used as the second polymer, sodium dodecyl sulfate (SDS) is used as the surfactant, and glutaraldehyde is used as the crosslinking agent. ) Or a method for producing a bilayer scaffold using Ethyldiethylaminopropylcarbodiimide (EDC) will be described in detail with one embodiment of the present invention.

That is, according to one embodiment of the present invention, preparing a gelatin aqueous solution by stirring at 100 to 300 rpm at 60 to 70 ℃ so that the gelatin is completely dissolved in water; Adding and stirring polyvinyl alcohol (PVA) to the gelatin aqueous solution; Adding sodium dodecyl sulfate to the stirred reactant at a high temperature and high speed at 80 to 120 ° C. to 800 to 2000 rpm; Lyophilizing the stirred reaction at -80 ° C. for 24 to 48 hours to obtain a sponge; Dipping the sponge in a crosslinking agent selected from glutaraldehyde or EDC (Ethyldiethylaminopropylcarbodiimide) to crosslink the sponge; And lyophilizing the crosslinked reactant at -80 ° C to prepare a bilayer scaffold.

First, gelatin, which is a natural polymer, is put in water and stirred at 100 to 300 rpm at 60 to 70 ° C. to prepare a completely dissolved gelatin aqueous solution. The gelatin aqueous solution is preferably 1 to 99% by weight gelatin aqueous solution, preferably 2.5% by weight gelatin aqueous solution. If the gelatin content in the aqueous solution of gelatin is out of the above range, it may cause a problem of not visually confirming that the layer separation occurs.

And, if the stirring conditions are out of the gelatin aqueous solution is not uniformly dissolved, it may cause a problem that the layer separation does not occur.

Polyvinyl alcohol (PVA) is added to the gelatin aqueous solution and stirred to prepare a polymer blend solution such that PVA is contained in an amount of 1 to 99% by weight, preferably 10% by weight. If the PVA content is out of the above range, it may cause a problem of not visually confirming that delamination occurs.

Sodium dodecyl sulfate (SDS) is added to the stirred polymer blend solution at 800 to 2000 rpm at 80 to 120 ° C., followed by high temperature and high speed stirring. At this time, the SDS is added so as to contain 0.01 to 2% by weight based on the entire polymer blend solution. If the SDS content is out of the above range, problems such as failure to control the reaction of layer separation or difficulty in handling may be caused.

In addition, when the high temperature and high temperature stirring conditions are out, even layer separation may not occur uniformly, or problems such as inability to control the reaction of the layer separation may be caused.

The stirred reaction was lyophilized at −80 ° C. for 24 to 48 hours to obtain a sponge, and the sponge was immersed in a crosslinking agent selected from glutaraldehyde or EDC (Ethyldiethylaminopropylcarbodiimide) and crosslinked. In this case, the crosslinking agent is an aqueous solution containing 0.1 to 1% by weight of a crosslinking agent such as glutaraldehyde or EDC (Ethyldiethylaminopropylcarbodiimide), and crosslinking for 3 to 24 hours. If the content of the crosslinking agent is out of the above range, the crosslinking reaction may not occur to decompose in solution, or may cause problems in cell culture due to the residue of the crosslinking agent which does not occur.

The crosslinked reactant is lyophilized again at -80 ° C to prepare a bilayer scaffold.

According to the embodiment, unlike the conventional double-layer scaffold manufacturing method prepared by manufacturing and attaching the polymer scaffold respectively, there is no possibility of layer separation through a single process without the attachment process double layer scaffold as shown in Figs. The folds can be made simply.

In addition, as a result of culturing the cells using the bilayer scaffold, as shown in FIG. 4, after 1 day of cell division, the cells adhered to the scaffold and started to proliferate. The extracellular matrix (ECM) can be secreted to confirm that ECM is deposited in the scaffold.

Therefore, the bilayer scaffold according to the present invention can be widely used as a material for the delivery of drugs, cells, proteins and growth factors or artificial skin in the fields of tissue engineering, medicine, pharmacy and materials science.

That is, the cells can be cultured in a bilayer scaffold according to the preparation method and used for tissue regeneration. The cells may be any one or two or more selected from the group consisting of chondrocytes, bone cells and skin cells, and the cells may be co-cultured by culturing different cells in the upper and lower layers of the bilayer scaffold. It may be.

In particular, the bilayer scaffold according to the present invention can be applied to skin defects. In other words, the cells are cultured in a bottom layer scaffold made of natural polymer and used as a tissue regeneration layer. The drug is loaded into the top layer scaffold made of synthetic polymer and bacteria from outside during tissue regeneration. It can stop infection. The natural polymer scaffold is biodegraded and removed as tissue regeneration proceeds, and the synthetic polymer scaffold is removed when tissue regeneration is completed.

In addition, the bilayer scaffold according to the present invention may be co-cultured with two kinds of cells and applied to a portion where tissues are in contact with each other, such as bone and cartilage, to help regeneration at the same time. That is, culturing chondrocytes in a bottom layer scaffold made of a natural polymer, culturing bone cells in a top layer scaffold made of a synthetic polymer, and then the double layer scaffold in a part where the tissue is in contact. Apply to help tissue regeneration.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

Example 1 Fabrication of Double Layer Scaffolds

Gelatin aqueous solution was prepared at 2.5% by weight and stirred at 80 rpm at 300 rpm. After the gelatin was completely dissolved, polyvinyl alcohol (PVA) was added to 10 wt% and stirred. Sodium dodecyl sulfate was added to 0.2% by weight, and the mixture was stirred at 95 ° C. and 900 rpm at high temperature and high speed. The stirred solution was poured into a petri dish and lyophilized at −80 ° C. for at least 24 hours to obtain a sponge. The sponge was immersed in an aqueous solution containing 0.5 wt% of glutaraldehyde and crosslinked for at least 12 hours. Again, lyophilization at −80 ° C. yielded a bilayer scaffold.

The double layer scaffold thus prepared was imaged with a field emission scanning electron microscope (FE-SEM), S-4100, HITACHI, LTD, as shown in FIG. 2, and a field emission scanning electron microscope (FE-SEM), S- SEM images observed with 4100, HITACHI, LTD are shown in FIG. 3. In other words, the double-layer scaffold is completely integrally attached to confirm that it is a safe double-layer scaffold without separation.

Experimental Example 1 Performance Evaluation of the Double Layer Scaffold

After preparing the bilayer scaffold prepared in Example 1cm in diameter, 2X10 ⁴ CRL-2310 (ATCC Number), keratinocyte, human papillomavirus 16 (HPV-16) E6 / E7 transformed was dispensed into the bilayer scaffold. As a result of SEM observation after cell division, as shown in FIG. 4, after 1 day of cell division, cells adhered to the scaffold and started to proliferate. As shown in FIG. 5, after 5 days of cell division, cells were extracellular matrix (ECM). ) Can confirm that ECM is deposited in the scaffold.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (10)

Preparing a gelatin aqueous solution;
Adding and stirring polyvinyl alcohol (PVA) to the gelatin aqueous solution;
Adding sodium dodecyl sulfate (SDS) to the stirred reactant and stirring at 800 to 2000 rpm at a temperature of 80 to 120 ° C .;
Lyophilizing the stirred reactant to obtain a sponge;
Dipping the sponge in a crosslinking agent selected from glutaraldehyde or EDC (Ethyldiethylaminopropylcarbodiimide) to crosslink the sponge; And
Lyophilizing the crosslinked reactant again
Method of manufacturing a double layer scaffold by a single process comprising a. delete delete delete delete delete The method of claim 1, further comprising: preparing a gelatin aqueous solution by stirring at 60 to 70 rpm at 100 to 300 rpm to completely dissolve the gelatin in water; Adding and stirring polyvinyl alcohol (PVA) to the gelatin aqueous solution; Adding sodium dodecyl sulfate to the stirred reactant and stirring the solution at 80 to 120 ° C. at 800 to 2000 rpm; Lyophilizing the stirred reactant to obtain a sponge; Dipping the sponge in a crosslinking agent selected from glutaraldehyde or EDC (Ethyldiethylaminopropylcarbodiimide) to crosslink the sponge; And lyophilizing the crosslinked reactant again. A tissue regeneration method comprising culturing cells in a bilayer scaffold according to any one of claims 1 to 7. The method of claim 8, wherein the cells are any one or two or more selected from the group consisting of chondrocytes, bone cells, and skin cells. The method of claim 8, wherein the cells are co-cultured by culturing different cells in the upper and lower layers of the bilayer scaffold.

Images