KR101617434B1 - Method for manufacturing multilayered scaffold for cartilage using biodegradable biopolymers - Google Patents

Abstract

The present invention relates to a method for producing a multilayered cartilage scaffold using an advanced biodegradable biopolymer, and more particularly, to a method for producing a cartilage scaffold comprising a biocompatible polycaprololactone (PCL), a sodium chloride used as a pore- To provide a gradient, whereby a cartilage support having a double layer having excellent physical properties (hard surface and smooth surface) can be produced. In addition, by controlling the size and shape of the saline system as a pore forming agent and the shape of the molding die, the membrane type of the cartilage support can be manufactured, and the material and mechanical characteristics can be easily controlled have.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for manufacturing a multilayered cartilage support using biodegradable biopolymers,

The present invention relates to a method for producing a multilayered cartilage support using an advanced biodegradable biopolymer.

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, a tissue of a human or an animal is taken, cells are separated from the tissue, and the cells are cultured on a support (scaffold) to prepare a cell-support complex, .

Tissue engineering techniques have been applied to almost all organs in human body such as artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessel and artificial muscle. To optimize the regeneration of such living tissues and organs, basically, Should be provided. In general, once a cartilage tissue of the vertebral joint is damaged, it is not regenerated normally in vivo.

Basically, the cartilage support should be non-toxic, have excellent mechanical properties, and be porous. In this case, the non-toxic refers to the absence of blood coagulation or inflammation reaction after cell-support complex and the support alone are transplanted into the living tissue, and the mechanical properties of the support are sufficient to support the growth of cells. Porosity not only allows cells to adhere well to the support but also ensures sufficient space between the cells and cells to supply oxygen and nutrients due to the diffusion of body fluids and to facilitate the formation of new blood vessels, It is a structure that can be done.

Cells are cultured in a two-dimensional manner. To cultivate them in the form of tissues or organs, a three-dimensional support is required. These supports have numerous pores, so cells must be able to adhere to the inside and outside, and have an open structure to feed the nutrients needed for cell growth and to release waste products. That is, a porous three-dimensional support is required.

Accordingly, as the supporter satisfying the above-mentioned basic requirements, it is preferable to be similar to the extracellular matrix in the animal body, and a method of producing a supporter having the same porosity as the extracellular matrix has been actively studied. Representative methods include particulate leaching, emulsion freeze-drying, high pressure gas expansion, phase separation, and electrospinning.

The particle leaching method is a method of forming pores by mixing salt particles which are not dissolved in a solution in which a biocompatible polymer is dissolved in an organic solvent, preparing a casting, removing the solvent, and removing salt particles by using water. However, the particle leaching method requires a large amount of salt to be used, and a method of controlling the pore size by controlling the size of the salt is adopted, so that there is a problem that the cells are damaged due to the remaining salt salt or rough shape.

US Patent Publication No. 5,681,873 (Oct. 28, 1997)

It is an object of the present invention to provide a cartilage support using a biodegradable biopolymer.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a method for producing a biodegradable biodegradable polymeric cartilage support having a multi-layer structure comprising the steps of:

a) dissolving the biopolymer in dichloromethane to prepare a biopolymer solution;

b) mixing the pore forming agent with the biopolymer solution prepared in step a);

c) placing the biopolymer solution in which the pore-forming agent is mixed in the step b) into a mold to obtain a molding agent; And

d) drying the molding agent at 0 占 폚 to 40 占 폚.

In one embodiment of the present invention, the method further comprises a step of washing the molding agent dried in the step d), followed by drying at 0 캜 to 40 캜.

In another embodiment of the present invention, the biopolymer of step a) is a biodegradable polymer.

In still another embodiment of the present invention, the biopolymer is selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), poly-epsilon -caprolactone (PCL) Poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymers, polyvinyl alcohols, polyethylene glycols, polyurethanes, polyacrylic acid, poly-N-isopropylacrylamide, ≪ / RTI >

In yet another embodiment of the present invention, the pore-former is characterized in that it is selected from the group consisting of crystalline salts, crystalline hydroxides, and water-soluble polysaccharides.

In another embodiment of the present invention, the crystalline salts are characterized in that they are selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride.

In another embodiment of the present invention, the crystalline hydroxide is characterized in that it is selected from the group consisting of sodium hydroxide and potassium hydroxide.

In another embodiment of the present invention, the water-soluble polysaccharide is characterized in that it is selected from the group consisting of sugar and starch.

In still another embodiment of the present invention, the pore-forming agent is used in an amount of 0.1 to 10 times that of the biopolymer.

In addition, the present invention provides a biodegradable bio-polymeric cartilage support having a multilayer structure prepared by the above method.

In one embodiment of the present invention, the support has a pore size of 100 to 500 μm and a porosity of 80 to 95%.

In another embodiment of the present invention, the porous biodegradable polymeric support is characterized by having a compressive strength of 10 Mpa to 100 Mpa.

According to the present invention, it is possible to produce a multilayered cartilage support according to a concentration gradient by controlling the composition ratio using salt-based sodium chloride, which is a biopolymer, polycaprolactone (PCL) used as a pore forming agent in a salt leaching process.

In addition, in the salt leaching action step, the shape, material and mechanical characteristics of the membrane type of the cartilage support can be easily controlled by adjusting the size, shape, and shape of the mold, which is a pore forming agent.

The present invention can obtain a desired cartilage support having a desired pore size and porosity and a good interconnection of the space through a simple manufacturing process. In addition, the present invention greatly reduces the manufacturing time of the cartilage scaffold and facilitates mass production, thereby improving the productivity and economy of the scaffold manufacturing industry. In addition, cartilage scaffolds having excellent effects of inducing cell proliferation, differentiation, and adhesion can be produced.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an electron micrograph of the naked eye and the structure of a support prepared by the method of Example 1. Fig.
Fig. 2 is a photograph schematically illustrating the manufacturing method of Example 1. Fig.
Figure 3 is a cartilage support made in the form of a membrane.
4 is an electron micrograph of the support structure produced.
Figure 5 is an electron micrograph of cells where cells migrate into the support and grow inside.
6 is a graph showing compressive strength and tensile strength of a support.
FIG. 7 is a photograph showing the cytotoxicity of the prepared support and the support alone when cultured with the cells.

The present invention relates to a method for producing a cartilage scaffold having a multilayer structure using a biodegradable biopolymer.

In the tissue engineering technique, in order for artificial tissues such as artificial skin, artificial bone, and artificial cartilage to be implanted in the living body, the artificial tissues must be well attached to the support and the physical properties of the support itself must be excellent. It must be excellent.

In the case of the particle leaching method, which has been used for the production of a conventional support for such a support, the amount of salt must be large and it is difficult to control the shape and size of the pores.

Therefore, the present inventors paid attention to the salt leaching process and found that the size and shape of the pores of the support can be controlled by controlling the composition ratio of the biopolymer and the salt as the pore-forming agent, and that the physical properties of the support are also excellent, .

That is, the present invention can provide a method for producing a biodegradable biodegradable polymeric cartilage support having a multilayer structure.

Hereinafter, the present invention will be described in detail by steps.

In the method for producing a cartilage scaffold of the present invention, step a) is a step of dissolving a solid polymer in dichloromethane to obtain a biopolymer solution.

The biopolymer may be a biodegradable polymer. Examples of the biodegradable polymer include polyglycolide, polylactides, polycaprolactones, polytrimethylenecarbonates, poly-hydroxy-butyrates, poly But are not limited to, polyhydroxyvalerates, polydioxanones, polyorthoesters, polycarbonates, polytyrosinecarbonates, polyorthocarbonates, polyalkylene oxalates, polyalkylene oxides, oxalates, polyalkylene succinates, poly (malic acid), poly (maleic-anhydride), polypeptides, polyphedipeptides (e.g., polydepsipeptides, polyvinyl alcohols, polyesteramides, polyamides, polyanhydrides, polyurethes, polyurethanes, anis, polyphosphazenes, polycyanoacrylate, polyfumarates, poly (amino acid), modified polysaccharides, modified proteins, and the like. And polymer mixtures thereof. Among these, polyglycolide, poly (L-lactide-co-glycolide) (poly (L-lactide-co-glycolide) ), Poly (D, L-lactide-co-glycolide), poly (L-lactide) D, L-lactide) (poly (L-lactide)), poly (L-lactide-co-D, L-lactide) , Polycaprolactone, poly (L-lactide-co-caprolactone), poly (D, L-lactide-co-caprolactone (D, L-lactide-co-caprolactone), polytrimethylenecarbonate, poly (L-lactide-co- Poly (L-lactide-co-trimethylenecarbonate), poly (D, L-lactide-co-trimethylene carbonate) Polydioxanone, copolymers thereof, ternary polymers thereof, polymer blends thereof, and the like can be used. Most preferably, polycaprolactones are used as in the embodiment of the present invention. But is not limited thereto.

The polymer content in the biopolymer solution according to step a) of the present invention is preferably 5 to 40% by weight, more preferably 10 to 20% by weight. This is because the paste phase of the support increases as the content of the biopolymer used as the support increases. This can significantly change the material and mechanical properties by changing the biopolymer content.

The biopolymer is dissolved in dichloromethane in an amount of 10 to 50 wt%, which changes the material and mechanical properties of the biopolymer after the salt leaching process. In addition, it is possible to reduce the contamination degree of the solvent by dissolving the solid biomolecule and the liquid dichloromethane in the liquid phase to 10 to 50 wt%, and the mixing with the salt as the pore forming agent gives the flowability of the biopolymer and rotation of the mixture, It is possible.

In the method for producing a cartilage scaffold of the present invention, step (b) is a step of adding a pore-forming agent to the biopolymer solution prepared in step (a), and dispersing and mixing the pore-forming agent evenly.

In the present invention, salts used as a pore-forming agent are uniformly dissolved in a solution of a biopolymer and supplied to a biopolymer mixture, whereby a porosity similar to that of a human bone structure is obtained This method is used to obtain a cartilage scaffold.

In addition, since it is possible to control the size of the salt, it is possible to manufacture a support having various sizes of pores and it has an advantage of being able to complement physical and mechanical forces required at specific sites.

As the pore former, salt-based sodium chloride or a pharmaceutically acceptable salt thereof may be used.

The pore-forming agent is preferably added to the biopolymer in an amount of 1 to 5 times. Here, it is also possible to manufacture supports having different structures or physical properties depending on the salt concentration. A support with a triple layer is made and a support with a double layer is produced when salt is added in a large amount. It is also possible to control the porosity by salt. It is possible to prepare a support having a porosity of 70% to 80% at the addition of less than 50%, 2 times at 5 times, and 90% or more at the addition of 5 times.

In addition, spherical sodium chloride having a particle size of 100 탆 or less, 200 탆 or 300 탆 can be used to obtain a final broth having a shape suitable for the pore size and osseointegration ability of the human bone structure and free of contaminants.

In the method for producing a cartilage scaffold of the present invention, in step c), a biopolymer solution mixed with a pore-forming agent is formed into a mold by molding the pore-forming agent in step b).

The mold may be a Teflon mold, and the mold may be 12 mm in height and 10 mm in height, but is not limited thereto.

At this time, depending on the size and shape of the mold (mold), the bone graft material, the bone plate, and various implants can be applied.

In the method for producing a cartilage scaffold of the present invention, step d) is a step of drying the molding agent.

The drying can be carried out at room temperature and at low temperature, preferably at 0 캜 to 40 캜, and most preferably at 4 캜 to 20 캜.

The method may further include a step of washing the molding agent dried in the step d) and then drying the molding agent again. The drying can also be carried out at room temperature and at a low temperature, preferably at 0 ° C to 40 ° C, and most preferably at 4 ° C to 20 ° C. Through this washing step, salts and impurities present in the support can be removed.

Through the washing and drying steps, salts added to the biodegradable biopolymer formed body can be removed to form pores and remove impurities. In this case, as the solvent for washing with water, tertiary distilled water can be preferably used, and when it is washed with 70% ethanol, impurities of the support may be removed more easily, but it is not limited thereto.

In addition, the cartilage scaffold prepared by the method of the present invention binds cartilage cells or bone cells, collagen, alginate, or peptide differentiated from human mesenchymatous stem cells or human mesenchymal stem cells to form a supporter having cell adhesion or drug delivery ability Can be manufactured. The collagen is preferably collagen type I, but is not limited thereto.

In addition, the support of the present invention may have a pore size of 100 to 500 μm, and may have a compressive strength of 10 MPa to 100 MPa, but is not limited thereto.

Accordingly, the present invention can produce a cartilage support having a multi-layered pore structure by mixing the biopolymer PCL and salts by controlling the weight ratio and drying at a room temperature and a low temperature by a salt leaching action process. The material, mechanical properties and biodegradability A controlled high-performance biodegradable biodegradable polymeric cartilage support can be obtained.

Hereinafter, embodiments of the present invention will be described in detail. It should be noted, however, that the following examples are illustrative of the present invention and that the present invention is not limited to the following examples.

[ Example ]

Example  One. Mold  Rounded PCL / Salt  By concentration Cartilage scaffold  Produce( sample  1, 2, 3, 4, 5, 6, 7, 8, 9)

The biodegradable biopolymer used in the preparation of the support is uniformly dissolved and dispersed in polycarbonate (PCL, 0.5 g, 1 g, 2 g) in dichloromethane (10 ml). NaCl (1 g, 2.5 g, 5 g, powder phase) as a pore-forming agent was added to the polymer material in the solution state, and the mixture was homogeneously dispersed. The mixture was injected into a mold and dried at room temperature and low temperature (4 ° C to -20 ° C), washed with water in tertiary distilled water and then dried in a dry oven at 37 ° C to obtain a multi-layered support. The composition of each of the polycaprolactone and pore-forming agent is shown in Table 1 below.

Comparative Example . Mold  Rounded PCL / Salt  By concentration Cartilage scaffold  Produce( Comparative Example  1, 2, 3)

The biodegradable biopolymer used for the support was prepared by uniformly dissolving and dispersing polycaprolactone (PCL, 0.5, 1, 2 g) in dichloromethane (10 ml). Then, the dispersed solution was injected into a molding frame immediately without adding a pore-forming agent and dried at room temperature and low temperature (4 to 20 ° C), washed with water of tertiary distilled water and then dried in a dry oven at 37 ° C to obtain a multi- A product was obtained.

FIG. 2 shows a manufacturing process chart of Example 1 and Comparative Example. SEM images of the obtained cartilage scaffold of the multi-layered structure are shown in FIG. 3. The characteristics of the initial materials used in Example 1 and Comparative Example, The ratio of the ratio and the final support is shown in Tables 1, 2 and 3. Therefore, it is considered that the difference in strength occurs depending on the concentration, so that it can be selected when the support is made according to the pathology.

Usage Configuration shape Size size PCL Template C ₆ H ₁₀ O ₂ elegance - Salt (NaCl) Porogen NaCl Powder ≤200 μm Dichloromethane menstruum CH ₂ Cl ₂ Liquid phase -

PCL: NaCl (%) PCL (g) NaCl (g) Dichloromethane Comparative Example 1 5: 0 0.5 0




10 mL Sample 1 5:10 0.5 One Sample 2 5:25 0.5 2.5 Sample 3 5:50 0.5 5 Comparative Example 2 10: 0 One 0 Sample 4 10:10 One One Sample 5 10:25 One 2.5 Sample 6 10:50 One 5 Comparative Example 3 20: 0 2 0 Sample 7 20:10 2 One Sample 8 20:25 2 2.5 Sample 9 20:50 2 5

PCL concentration 5% PCL 10% PCL 20% PCL Salt PCL Salt PCL Salt PCL 0 0.1 3.9 0.05 8.7 0.3 11.8 One 2.25 4.75 8.3 8.2 7.8 14.55 2.5 2.05 4.05 22.4 8.05 21.95 11.4 5 16.1 4.55 44.65 6.65 47.2 13.35

Example  2. Mold  Used Membrane  Type support

The biodegradable biopolymer used in the preparation of the support was prepared by uniformly dissolving and dispersing polycaprolactone (PCL 2 g) in dichloromethane (10 ml), adding 5 g of pore forming agent (5 g, ) Were added and dispersed uniformly. Then, the mixture is poured into a Teflon mold, and left at room temperature for 1 day to obtain a double layer support. As shown in Fig. 1, this support is characterized by having a sponge type having a soft side at the lower side and a type having a hard side at the upper side.

Example  3. Manufactured Cartilage  Check section

Analysis was performed using a scanning electron microscope (FE-SEM, JMS-6335F, JEOL, Japan) in order to obtain sample surface information of the bone supports of Examples 1 to 2 and Comparative Examples of the present invention. As shown in FIG. 1, the cross section of the prepared biodegradable support was analyzed and it was observed that pores were formed. In addition, it was confirmed that the salt leaching action through the washing process was completed to form pores.

4, closed pores were formed in the comparative example (left side), whereas open pores (interconnected pores) were formed not in the closed pores in the embodiment, It was confirmed that the active deposition and adherence of the cartilage scaffold of the present invention would be effective in relation to the behavior behavior and hydrophilicity of the cartilage support of the present invention.

Example  4. Manufactured Cartilage  cell Attachment  Confirm

FIG. 5 shows the results of experiments to confirm that cells adhere well to the support of the present invention. Cells used in this example were tested for their ability to adhere using NIH3T3. NIH3T3 was plated on a 24-well plate at 1 × 10 ⁵ / ml, incubated at 37 ° C for one day, and incubated for 7 days with 70% alcohol-disrupted support. The medium was replaced at 2-day intervals, fixed at 4% formalin on day 7, and stained with SRB (sulforhodamin B) to confirm cell adhesion to the scaffold. 5, it was confirmed that the structure of the support of the present invention is excellent in adhesion of cells. It was confirmed that the cells migrated to the supporter (a), or that the cells did not grow (b).

Example  5. Identification of physical properties of the prepared support

In addition, the physical properties of the support prepared by the FITI test institute were analyzed and shown in FIG. As shown in FIG. 6, the compressive strength (Instron Mechanical Tester) and tensile strength of the cartilage scaffold of Comparative Example 1 and Examples 1 and 2 were analyzed. As a result, the extrusion strength was 50 Mpa or more and the tensile strength was 1.3 The tensile strength of Mpa was shown.

Example  6. Cytotoxicity of the prepared support

The supernatant prepared in Examples 1 and 2 was placed on a 24-well plate of NIH3T3 cells at 1 × 10 ⁴ / ml and cultured for 1 day. The eluate was added at a concentration of 8 and 16%, and in the case of the support, The cells were cultured for 3 days and cytotoxicity was observed. SRB staining was used to facilitate observation. As shown in Fig. 7, it was observed that addition of the eluent (a) and the support (b) did not affect the cell growth.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the embodiments described above are in all respects illustrative and not restrictive.

Claims (12)

a) dissolving the biopolymer in dichloromethane to prepare a biopolymer solution;
b) mixing the pore forming agent with the biopolymer solution prepared in step a);
c) placing the biopolymer solution in which the pore-forming agent is mixed in the step b) into a mold to obtain a molding agent; And
d) drying the molding agent at 0 캜 to 40 캜,
The pore-forming agent is mixed 1 to 5 times by weight of the biopolymer
(METHOD FOR MANUFACTURING BIOLOGICAL SOLUBLE BIOMEDICAL HYBRID CARBON SUPPORT OF MULTILAYER STRUCTURE)
The method according to claim 1,
Further comprising the step of washing the molding agent dried in the step (d), followed by drying at 0 캜 to 40 캜.
The method according to claim 1,
Wherein the biopolymer in step a) is a biodegradable polymer.
The method of claim 3,
The biopolymer may be selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), poly-epsilon -caprolactone (PCL), polyanhydride, polyorthoesters, (Ethylene oxide) -poly (ethylene oxide) -poly (ethylene oxide) copolymer, wherein the polyol is selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, Lt; / RTI >
The method according to claim 1,
Wherein the pore-former is selected from the group consisting of crystalline salts, crystalline hydroxides, and water-soluble polysaccharides.
6. The method of claim 5,
Wherein the crystalline salts are selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, and magnesium chloride.
6. The method of claim 5,
Wherein the crystalline hydroxide is selected from the group consisting of sodium hydroxide, and potassium hydroxide.
6. The method of claim 5,
Wherein said water soluble polysaccharide is selected from the group consisting of sugar and starch.
delete The biodegradable bio-polymeric cartilage scaffold of multilayer structure prepared in claim 1.
11. The method of claim 10,
The biodegradable bio-polymeric cartilage scaffold of the multilayer structure, wherein the support has a pore size of 100 μm to 500 μm and a porosity of 80% to 95%.
11. The method of claim 10,
Wherein the porous biodegradable polymeric scaffold has a compressive strength of 10 Mpa to 100 Mpa.

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