WO2019112184A1

WO2019112184A1 - Three-dimensional fiber-type scaffold

Info

Publication number: WO2019112184A1
Application number: PCT/KR2018/013216
Authority: WO
Inventors: 김태희; 임정남; 도성준; 김채화; 김윤진
Original assignee: 한국생산기술연구원
Priority date: 2017-12-05
Filing date: 2018-11-01
Publication date: 2019-06-13
Also published as: KR20190066546A; KR102233499B1

Abstract

Provided is a three-dimensional fiber-type scaffold having a plurality of pores, wherein the scaffold includes a fiber structure having two types of fibers having different biodegradation rates, wherein the two types of fibers having different biodegradation rates comprise a biodegradable first fiber and a biodegradable second fiber which has a slower biodegradation rate than the first fiber, or comprise a biodegradable first fiber and a non-biodegradable second fiber.

Description

Three-dimensional fiber type scaffold

The present invention relates to a three-dimensional fiber-type scaffold. More specifically, the present invention relates to a three-dimensional fiber-type scaffold which can be molded into a desired shape and is effective for cell and tissue regeneration, to be.

This application is based upon and claims the benefit of Korean Patent Application No. 10-2017-0165617, filed December 5, 2017, the entire contents of which are incorporated herein by reference.

Numerous surgical operations are being undertaken every day worldwide to replace, rebuild or restore the tissue of a human body damaged by disease or injury. Use tissues from other parts of the patient for the purpose of replacing tissue damage or implant tissue of another person's body. These treatments can prolong life, but the transplantation of the tissue is painful, requires a lot of expenses, has infection problems, and has difficulties in securing organ transplants. Furthermore, there may be rejection by the patient's immune system.

In the field of tissue engineering, which is used almost synonymous with regenerative medicine, cells or tissues biologically obtained from a patient's body are cultivated in a scaffold to apply the damaged part to the damaged area, thereby improving or maintaining the function of the tissue, The goal is to ensure that. Tissue engineering is defined as the use of the principles and methods of engineering and life sciences to understand the relationship between the structure and function of mammalian tissues that are normal or pathological and to develop biological substitutes for the recovery and improvement of tissue function .

Tissue engineering uses a porous three-dimensional scaffold for tissue or organ regeneration, and the scaffold is used as a template for tissue formation. Such a scaffold should have responsive properties to various types of mechanical and chemical stimuli that include cells or growth factors, have a function in response to bio-physical stimuli in the form of a bioreactor, or are applied to cells.

On the other hand, existing tissue regeneration scaffolds are easy to support cells, but they are difficult to be molded into a desired shape or shape, or have physical properties suitable for the tissue, and thus, application to the actual tissue has been limited. In addition, existing scaffolds are disadvantageous for cell proliferation due to the structure in which the internal pores are not connected to each other, and it is difficult to control the biodegradation rate of the scaffold to match the regeneration rate of the tissue, .

In addition, in conventional scaffolds, it is possible to apply only to a form in which cells are seeded and cultured and then inserted into a human body together with a scaffold. When a large amount of cells must be cultured in a bioreactor through a scaffold, It was not easy to separate cells from the scaffold and collect them.

Therefore, the scaffold containing the cells can be cultured out of the living body through a bioreactor or the like and the partially synthesized tissue can be easily collected from the scaffold to be implanted on the wound site, or placed directly on the wound site, There is a great demand for development of a scaffold which can be applied to regenerate a tissue without limitation and which can be formed into a desired shape or shape.

A problem to be solved by the present invention is to provide a three-dimensional fiber-type scaffold which can be freely formed into a desired form, is highly effective for cell adhesion, proliferation and regeneration, and is easy to collect cells or tissues regenerated after cell or tissue regeneration will be.

In order to solve this problem, according to one aspect of the present invention, there is provided a three-dimensional fiber-type scaffold of the following embodiment.

In a first embodiment,

A three-dimensional fiber-type scaffold having a plurality of pores,

Wherein the scaffold includes a fiber structure having two kinds of fibers having different biodegradation rates,

Wherein the two types of fibers having different biodegradation rates include a biodegradable first fiber and a biodegradable second fiber having a slower biodegradation rate than the first fiber or a biodegradable first fiber and a non biodegradable second fiber To a three-dimensional fiber-type scaffold.

The second embodiment, in the first embodiment,

Dimensional fibrous scaffold in which the biodegradable first fiber has a melting point higher than that of the biodegradable second fiber or the non-biodegradable second fiber, or has no melting point.

The third embodiment, in the first or second embodiment,

Dimensional scaffold in which the average size of the pores of the scaffold increases with time.

The fourth embodiment is, in any one of the first to third embodiments,

Wherein the biodegradable first fibers and the biodegradable second fibers are selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid, polyhydroxybutyric acid- Valeric acid copolymer, collagen, hyaluronic acid, cellulose oxide, chitosan, chitin, gelatin, silk fibroin or a mixture of two or more thereof.

The fifth embodiment is, in any one of the first through fourth embodiments,

Wherein the non-biodegradable second fiber comprises a polyolefin, a polyester, a polyamide, or a mixture of two or more thereof.

The sixth embodiment is, in any one of the first through fifth embodiments,

Wherein the biodegradable first fiber comprises polyglycolic acid (PGA), a copolymer of polylactic acid-glycolic acid (PLGA), or a mixture thereof, the biodegradable second fiber comprises polylactic acid, 1 fiber is selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid, polyhydroxybutyric acid-valeric acid copolymer, collagen, hyaluronic acid, Wherein the non-biodegradable second fibers are selected from the group consisting of polypropylene, polyethylene, polyethylene terephthalate (PET), polyethylene terephthalate copolymer, polybutylene Terephthalate (PBT), polytrimethylene terephthalate (PTT), polycyclohexanedimethanol terephthalate (PCT), and poly Dimensional fibrous scaffold comprising tilene naphthalate (PEN), nylon 6,

nylon

6,6, nylon 4 and

nylon

4,6 or a mixture of two or more thereof.

The seventh embodiment is, in any one of the first through sixth embodiments,

Said scaffold being in the form of a nonwoven; Fabric type; Knitted form; Fiber bundle type; A cylindrical shape having a fiber bundle and a tube into which the fiber bundle is inserted, or a three-dimensional fiber type scaffold that is a mixture of two or more thereof.

The eighth embodiment is, in any one of the first through seventh embodiments,

Dimensional fibrous scaffold in which the biodegradation rate can be controlled by gamma irradiation.

The ninth embodiment is, in any one of the first through eighth embodiments,

Dimensional fibrous scaffold in which the fibrous structure further comprises one or more fibers having different biodegradation rates.

The three-dimensional fiber-type scaffold according to an embodiment of the present invention can control the rate of biodegradation by complexing a fiber material having a different biodegradation rate so that the fiber material having a slow biodegradation rate acts as a binder or a skeleton, It is possible to provide a scaffold having a structure capable of maintaining a scaffold shape, capable of forming a thermoplastic material into a desired shape by thermoforming, and exhibiting excellent cell adhesion and cell proliferation.

In addition, the three-dimensional fiber-type scaffold according to an embodiment of the present invention has a scaffold pore size gradually increased according to the degree of cell culture and proliferation as a result of complexing fibrous materials having different biodegradation rates, The cultured cells can be easily separated (collected) from the scaffold.

Further, the fibrous scaffold according to an embodiment of the present invention is easy to be manufactured in a large amount, is convenient to be inserted into a necessary portion due to the flexibility of the fiber, is easy to modify the surface so as to increase cell affinity , And internal pores are connected to each other, which can be very advantageous for cell attachment and growth.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate preferred embodiments of the invention and, together with the description of the invention given below, serve to further the understanding of the technical idea of the invention, And should not be construed as limiting.

Figure 1 is a photograph of a vial undergoing a biodegradation test on the scaffolds prepared in Examples 1-3.

FIG. 2 is a graph showing changes in average pore size of the scaffold prepared in Examples 1 to 3 with time. FIG.

3A and 3B are SEM photographs showing the average pore size and surface change of the scaffold prepared in Examples 1 to 3 with time.

FIGS. 4 to 6 are graphs showing the results of biodegradation experiments according to the irradiation dose of gamma rays of the scaffolds prepared in Examples 1 to 3. FIG.

7 is an SEM photograph showing the results of biodegradation experiments of the scaffold prepared in Example 3 after 12 days of gamma irradiation dose of 0 kGy.

8 is an SEM photograph showing the result of biodegradation test of the scaffold prepared in Example 3 after the lapse of 12 days from the irradiation dose of 40 kGy of the gamma ray.

9 is a graph showing the results of analysis of the initial cell adhesion rate of the scaffolds prepared in Examples 1 to 3. Fig.

10 is a graph showing cell growth analysis results of the scaffolds prepared in Examples 1 to 3. FIG.

11 is a schematic view of a manufacturing process of a cylindrical scaffold according to the fourth embodiment.

12 to 14 are graphs showing the results of analysis of the biodegradation rate according to the irradiation dose of gamma rays of the scaffolds prepared in Examples 4 to 6. FIG.

15 to 17 are photographs showing SEM observation results of gamma ray irradiation amounts OkGy, 30 kGy, and 50 kGy of the scaffolds prepared in Examples 4 to 6. FIG.

[Correction according to Rule 91 of the Regulations of 08.01.2019]
18 is a graph showing the results of cell growth analysis according to the variation of irradiation dose of the scaffold in Example 3 and Comparative Example 1. Fig.
FIG. 19 is a graph showing the results obtained by taking a sample on the 12th day and the 33rd day after incubation of the sample prepared in Example 3, completely drying the sample in a 40 ° C convection oven, freezing it in liquid nitrogen and heating it for 150 seconds with an ion coater (E-1045) Coated, and the surface was observed using a field emission scanning microscope (FESEM, SU 8010).

Hereinafter, the present invention will be described in detail. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined.

Therefore, the configurations shown in the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the technical ideas of the present invention, so that various equivalents And variations are possible.

According to an aspect of the present invention, there is provided a three-dimensional fiber-type scaffold having a plurality of pores, wherein the scaffold includes a fiber structure having two types of fibers having different biodegradation rates, Wherein the fiber comprises a biodegradable first fiber and a biodegradable second fiber having a slower biodegradation rate than the first fiber, or a three-dimensional fiber type scaffold comprising a biodegradable first fiber and a non-biodegradable second fiber / RTI >

As compared with the existing tissue regeneration scaffold, the three-dimensional fiber type scaffold according to an embodiment of the present invention is a composite material in which fibers having different biodegradation characteristics are combined or thermally deformable are combined together, Can be molded into a desired shape.

Conventional scaffolds are easy to support cells, but they are difficult to be molded into a desired shape or shape, or to have a proper physical property for a tissue, and thus, application to a real tissue has been limited. However, according to one embodiment of the present invention, the biodegradable first fiber may have a higher melting point than the biodegradable second fiber or the non-biodegradable second fiber. The biodegradable second fiber or the non-biodegradable second fiber having a relatively low melting point can be molded into a desired shape through heat treatment during the production of the scaffold. According to one embodiment of the present invention, the biodegradable first fiber may be pyrolyzed immediately without melting point.

In addition, since conventional scaffolds have a structure in which internal pores are not connected to each other, cell proliferation is difficult, and it is not easy to control the biodegradation rate of the scaffold to match the regeneration speed of the tissue. However, the scaffold according to an embodiment of the present invention is characterized in that even if the biodegradable first fiber having a large biodegradation rate is decomposed or cut first after the cells are seeded, the biodegradable rate is slow The biodegradable second fiber or the non-biodegradable non-biodegradable second fiber serves as a skeleton capable of stably growing cells while maintaining the morphology of the scaffold. In addition, as the biodegradable first fiber having a large biodegradable rate is decomposed, the average size of the pores of the scaffold may increase with time.

The scaffold according to an embodiment of the present invention was prepared as a sample cut into a size of 2.5 x 2.5 cm and 100 ml of a phosphate buffered saline (PBS) (pH 7.4) solution was added to a 100 ml vial, After the sample was taken out in time, the sample was completely dried in a convection oven at 40 ° C., and then a capillary flow porometer (CFP-A) was placed in a shaking bath (60 ° C., 80 rpm) 1200AEL, Porous Materials Inc.), the pore size may increase from the start of observation of the biodegradation rate, for example, from 9 to 15 days.

As the average size of the pores of the scaffold according to an embodiment of the present invention increases, a sufficient space can be secured in the inside of the scaffold, and the cells seeded on the scaffold can exhibit an excellent effect for stably growing and propagating. Furthermore, there is an advantage in that the cells can be easily separated (collected) from the scaffold through the enlarged pores.

In the present invention, the biodegradable fiber refers to a fiber made of a polymer capable of being degraded by water or an internal degradation enzyme, and the biodegradation rate means a degree of speed at which the fiber is decomposed with time . In one embodiment of the present invention, the biodegradation rate of such a biodegradable fiber or a scaffold having such a biodegradable fiber can be confirmed by measuring a pH which is changed by an acid generated upon hydrolysis of the biodegradable fiber. For example, the rate of biodegradation can be compared based on the degree of change in pH that decreases over time relative to the initial time. Specifically, the sample was cut into a predetermined size (for example, 2.5 × 2.5 cm), and 50 ml of a phosphate buffered saline (PBS, pH 7.4) solution was added to a 50 ml Falcone tube. The sample was immersed in a 50 ml Falcone tube and shaken in a shaker incubator. (37 ° C, 1000 rpm), and the pH of the PBS is measured at each time point to confirm the biodegradation rate.

When the two kinds of fibers having different biodegradation rates include the biodegradable first fiber and the biodegradable second fiber having a slower biodegradation rate than the first fiber, the biodegradable first fiber and the biodegradable second fiber are biodegradable If the fibers are made of a biodegradable polymer material of which the speed of the fibers is different, the fiber can be selected and applied without limitation.

As the biodegradable first fibers and the biodegradable second fibers, fibers made of various biodegradable polymer materials can be applied. Non-limiting examples of the biodegradable first fibers include polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid Copolymers of polyhydroxybutyric acid and valeric acid, collagen, hyaluronic acid, cellulose oxide, chitosan, chitin, gelatin, silk fibroin or a mixture of two or more thereof And mixtures thereof. Here, the biodegradable first fibers and the biodegradable second fibers can be appropriately selected so that the difference in biodegradation rate can be obtained.

The non-biodegradable second fiber may be a fiber made of one or more kinds of polymer materials selected from various non-biodegradable synthetic fibers, and examples thereof include polyolefins, polyesters, polyamides, &Lt; / RTI > Specifically, the non-biodegradable second fibers may be selected from the group consisting of polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polycyclohexanedimethanol terephthalate (PCT) A phthalate (PEN); Polyamide-based polymers selected from nylon 6,

nylon

6,6, nylon 4, and

nylon

4,6; Or a polyolefin-based polymer selected from polyethylene or polypropylene, or a mixture of two or more thereof. For example, two or more of the polyester-based polymers may be selected as the mixture of the two or more, or a mixture of one or more of the polyester-based polymers and one of the polyolefin-based polymers may be respectively selected.

According to one embodiment of the present invention, the biodegradable first fiber comprises polyglycolic acid (PGA), a copolymer of polylactic acid-glycolic acid (PLGA), or a mixture of two or more thereof, May include polylactic acid.

When the two kinds of fibers having different biodegradation rates include the biodegradable first fiber and the non-biodegradable second fiber, for example, the first biodegradable fiber may be polylactic acid (PLA), polyglycolic acid , Copolymers of polycaprolactone, copolymers of polylactic acid-glycolic acid (PLGA), polyhydroxybutyric acid, polyhydroxyvaleric acid and polyhydroxybutyric acid-valeric acid, or a mixture of two or more thereof, Wherein the second fiber is selected from the group consisting of polypropylene, polyethylene, polyethylene terephthalate (PET), polyethylene terephthalate copolymer, polybutylene terephthalate (PBT), polytrimethylene terephthalate (PTT), polycyclohexanedimethanol terephthalate ) And polyethylene naphthalate (PEN), nylon 6,

nylon

6,6, nylon 4, and

nylon

4,6 or a mixture of two or more thereof .

According to an embodiment of the present invention, the weight ratio of the biodegradable first fiber and the biodegradable second fiber, the biodegradable first fiber, and the non-biodegradable second fiber among the two types of fibers having different biodegradation rates is 10:90 To 90:10, or 20:80 to 80:20, or 30:70 to 70:30.

In the case of polylactic acid-glycolic acid copolymers, lactide and glycolide may be copolymerized at various weight ratios, for example, at a weight ratio of 10:90 to 30:70.

According to an embodiment of the present invention, the scaffold is in the form of a nonwoven fabric; Fabric type; Knitted form; Fiber bundle type; A cylindrical shape having a fiber bundle and a tube into which the fiber bundle is inserted, or a mixture of two or more thereof.

The scaffold of the present invention can perform functions such as cell culture, cell delivery, or drug delivery using a space formed by the fibers contained in such a fiber structure.

According to an embodiment of the present invention, the fiber structure may further include one or more fibers having different biodegradation rates. For example, when the scaffold including the above-described fiber structure is a non-woven fabric, a woven fabric, a knitted fabric, a fiber bundle, or a cylinder, each of these fibers may have three kinds of fibers having different biodegradation rates, Fibers.

In particular, when the scaffold is in the form of a cylinder having a fiber bundle and a tube into which the fiber bundle is inserted, the fiber bundle may include two or more kinds of fibers having different biodegradation rates, The fiber bundle and the tube may include two or more kinds of fibers having different biodegradation rates from each other.

As used herein, a fiber, fiber form, or fiber refers to a group of linear filaments that are long, thin, and small in bending resistance so that they can bend, and such fibers may have micro- (Short cut fibers) cut into 0.1 to 40 mm, dry nonwoven short fibers (staple fibers) having a length of 10 to 130 mm, continuous fibers (long fibers, filaments) for fabrics and knitted fabrics, And the like.

Here, the nonwoven fabric refers to a nonwoven fabric in which the fibers are arranged in a parallel or non-uniform direction (uneven direction) without being subjected to a woven fabric process, and the fibers are mechanically entangled or thermally bonded, Means a kind of fiber structure obtained by joining together.

Such a nonwoven fabric may be manufactured by various methods such as wet (or sinking or super-knowledge), dry, spun lace, electrospinning, and the like.

In the dry nonwoven fabric, a thermal bonding nonwoven fabric prepared by mixing fibers having low melting point plasticity and igniting or dissolving them by heat, pressure, or the like, and bonding fiber structures, airborne (air laid nonwoven fabric, or a needle punching nonwoven fabric produced by physically combining a web with a fiber using a special needle, and is preferable in terms of improving the tensile strength, that is, the morphological stability of the nonwoven fabric. In the wet nonwoven fabric, the same process as that of the papermaking process is used. However, various fibers are used as raw materials but not as pulp. After the fibers are dispersed in an agate solvent, that is, a dispersion solution, the papermaking solvent is removed through a papermaking wire or a screen It is advantageous in that it can be made into a nonwoven fabric by drying and is preferable from the viewpoint of improvement of uniformity and can use short fibers which are relatively short compared to a dry process like microfibril.

The term "fabric" refers to a fabric structure fabricated by crossing fibers with warp yarns and weft yarns, and the knitted fabric refers to a fabric structure produced by continuous looping of one fiber, and is manufactured by various methods applicable in the art .

In addition, the pores of the scaffold refer to spaces formed by the fibers contained in the fiber structure constituting the scaffold, that is, pores formed between adjacent fibers or entangled fibers.

The size of the pores has a pore size of, for example, 1 to 150 mu m, particularly 5 to 50 mu m. At this time, when the pore size satisfies the above range, the cells and the drug can be improved in retention ability because the cells and the drug content that can be delivered in vivo can be appropriately controlled in favor of cell proliferation during cell culture.

According to one embodiment of the present invention, when the average pore size of the scaffold is about 15 days from the start of cell culture, the average pore size may be increased by about 1.2 to 3 times the average pore size of the initial scaffold have. This is because the biodegradable fibers constituting the scaffold are biodegraded or cut and the pores between the fibers are further expanded.

According to one embodiment of the present invention, the biodegradable first fiber, the biodegradable second fiber, and the non-biodegradable second fiber are each independently in the form of monofilament of 1 to 50 denier, or one strand of 0.5 to 4 The denier fibers may be tens or hundreds of strands of fibers that are spinnable in the form of multifilaments of 20 to 500 denier, or synthetic and natural staple fibers.

The cylinder shape having a fiber bundle and a tube into which the fiber bundle is inserted, which is a form of a scaffold according to an embodiment of the present invention, is characterized in that a multifilament twist yarn is inserted and fixed in a tubular circular- . Specifically, the tube may be formed of a non-biodegradable second fiber or a biodegradable second fiber having a slow biodegradation rate, and the fiber bundle inserted in the tube may be a biodegradable first fiber alone or a biodegradable first A mixture of the fiber and the biodegradable second fiber, or a mixture of the biodegradable first fiber and the non-biodegradable second fiber. Of course, additional fibers with different biodegradable rates may also be included independently in the tube or fiber bundle, respectively.

According to an embodiment of the present invention, as the fiber bundle to be inserted, a multi-filament false twist yarn secured in the connection of the inner space by the bulkiness characteristic in which the volume increase rate of 150 to 1000% Can be applied.

A cylindrical scaffold according to an embodiment of the present invention is characterized in that 1) a multifilament yarn made of a biodegradable polymer (or a non-biodegradable polymer) is put into a knot ring knitting machine to prepare a tubular circular piece, 2) Polymer (or two or more biodegradable polymers having different biodegradation rates, or biodegradable polymers and non-biodegradable polymers) may be spun into monofilaments or multifilament yarns by melt spinning or wet spinning and then spun into a biodegradable multifilament yarn 3) inserting the biodegradable multifilament false-twist yarn of step 2) into the tubular circular piece of step 1), and 4) inserting the inserted biodegradable multifilament false-twist yarn in an amount of about 10 to 50% Can be produced by stretching the false-twist yarn to give a bulky property at about 15 to 30%. (See Fig. 11)

According to an embodiment of the present invention, after the three-dimensional fiber-type scaffold is manufactured, further gamma-ray irradiation may be further performed. This gamma irradiation step can further improve the degree of biodegradation of the biodegradable first fiber constituting the three-dimensional fiber-type scaffold. As a result, the biodegradable first fiber and non-biodegradable In the case of containing the second fiber, the difference in the biodegradation rate between these two fibers can be made larger.

Furthermore, this gamma irradiation step may have the effect of sterilizing the three-dimensional fiber-type scaffold directly applied to the growing cells without using heat or chemicals. The gamma irradiation may be carried out at an irradiation dose of, for example, 1 to 100 kGy, particularly 5 to 70 kGy.

As described above, the three-dimensional fiber-type scaffold according to an embodiment of the present invention has a scaffold pore size gradually increased according to the degree of cell culture and proliferation as a result of complexing a fiber material having a different biodegradation rate, , And the cultured cells can be easily separated (collected) from the scaffold.

The degree of collection of the cultured cells, that is, the cell collection ratio (%), can be evaluated according to the following method:

"Tryptin (HyClone ™, 0.25% Trpsin) is added to the PBS in 10 ml of PBS by adding 2 ml of diluted trypsin solution into the scaffold sample by seeding the cells on a 24-well plate After incubation for 30 minutes at 37 ° C, 1 ml of medium was added per well, pipetted with a pipette to remove the cells attached to the scaffold, transferred to a tube and centrifuged at 1500 rpm The number of cells harvested relative to the number of cells grown in the scaffold is calculated and analyzed for cell collection rates. "

The three dimensional fiber scaffold according to an embodiment of the present invention may have a cell collection rate of 70% or more, 70% to 100%, or 80% to 100%. When the weight ratio of the biodegradable first fiber and the biodegradable second fiber, the biodegradable first fiber and the non-biodegradable second fiber among the two kinds of fibers having different biodegradation rates is 30:70 to 70:30 , &Lt; / RTI > 80% to 100%.

In the case where the three-dimensional fiber-type scaffold according to an embodiment of the present invention is a cylindrical shape having a fiber bundle and a tube into which the fiber bundle is inserted, the cells are grown while biodegradation occurs, And the tube, which is not an external structure, are separated naturally, and cell collection can be facilitated.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example 1 (Production of nonwoven fabric type scaffold)

PLGA fiber) (fineness: 2 denier, fiber length: 3 mm, melting point: 210 DEG C) 30 (lactic acid-glycolic acid copolymer (PLGA) (lactic acid: glycolic acid = 10:90 weight ratio) (Hereinafter referred to as " ES fiber ") composed of polypropylene (PP) (melting point: 160 DEG C) and a sheath made of polyethylene (PE) ) (Fineness: 3 denier, fiber length: 6 mm) was prepared.

The prepared fibers were first washed with 95% ethanol to remove the remaining emulsion, and then a wet nonwoven fabric was prepared using 100% distilled water as a dispersion medium. The prepared fiber mixture was sufficiently kneaded in an edible solution, and wet water was removed using a hand sheet former to prepare a wet nonwoven fabric. Thereafter, it was dried in a convection oven at 40 DEG C for 2 hours and thermally fused at 140 DEG C for 30 minutes to prepare a 200 g / m < ² > nonwoven fabric type scaffold composed of PLGA fibers and ES fibers at a weight ratio of 30:70.

Example 2 (Production of nonwoven fabric type scaffold)

Nonwoven fabric type scaffold was prepared in the same manner as in Example 1, except that PLGA fibers and ES fibers were mixed at a weight ratio of 50:50.

Example 3 (Production of nonwoven fabric type scaffold)

Nonwoven fabric type scaffold was prepared in the same manner as in Example 1, except that PLGA fibers and ES fibers were compounded at a weight ratio of 70:30.

In Examples 1 to 3, a nonwoven fabric constituting a scaffold can be formed into a desired shape by combining a PLGA fiber as a biodegradable material and a low melting point ES fiber to produce a scaffold made of a wet nonwoven fabric, It is possible to make collection convenient after cell culture.

Experimental Example 1 (Biodegradation test)

The samples prepared in Examples 1 to 3 were cut to a size of 2.5 x 2.5 cm and then 100 ml of a phosphate buffered saline (PBS) (pH 7.4) solution was added to 100 ml vials and immersed in a 100 ml vial, (60 ° C, 80 rpm), and the rate of biodegradation was observed at intervals of 3 days (see FIG. 1). The sample taken out over time was completely dried in a 40 ° C convection oven and then the average pore size was measured using a Capillary Flow Porometer (CFP-1200AEL, Porous Materials Inc.), frozen in liquid nitrogen, Coater, E-1045) coated with gold for 150 seconds, and the surface was observed using a field emission scanning microscope (FE-SEM, SU 8010). The change of the average pore size with time is shown in the graph of FIG. 2 and the SEM photograph of FIGS. 3A to 3B.

Referring to FIGS. 2, 3A, and 3B, the biodegradation test was conducted for about 15 days. The disrupted fibers were observed from about 6 days, and the decomposed form of the wet nonwoven fabric having a high PLGA fiber content And the average pore size was increased.

Experimental Example 2: Biodegradation Experiment with Gamma Irradiation

Each sample prepared in Examples 1 to 3 was irradiated with a dose of 10, 20, 30, 40, 50 kGy using a gamma ray irradiation equipment (MDS Nordion Inc C-188). Samples prepared according to the irradiation amount were cut into a size of 2.5 × 2.5 cm and 50 ml of a phosphate buffer saline (PBS, pH 7.4) solution was added to a 50 ml Falcone tube. The samples were immersed in a shaker incubator (37 ° C., 1000 rpm), and the pH of the PBS was measured at each time point to confirm the biodegradation rate. The results are shown in Fig. 4 to Fig.

[Correction according to Rule 91 of the Regulations of 08.01.2019]
On the 12th day and the 33rd day after the incubation of the sample prepared in Example 3, the sample was taken out and completely dried in a convection oven at 40 ° C, frozen in liquid nitrogen, and coated with an ion coater (E-1045) And the surface was observed using an electric field-type scanning microscope (FE-SEM, SU 8010). The observed results are shown in Fig. In addition, the sample prepared in Example 3 was taken out on the 12th day after incubation and observed with SEM. As a result, it was found in Fig. 7 that the dose of gamma irradiation was 0 kGy and in Fig. 7 that it was 40 kGy.

As shown in FIGS. 4 to 6, the biodegradation rate of the nonwoven fabric samples was increased with increasing gamma irradiation dose, and the faster the biodegradation rate, the faster the biodegradation rate was, Respectively.

Experimental Example 3: Initial cell adhesion rate analysis

NIH 3T3 (mouse embryonic fibroblast cell line) was prepared as a cell, and the cells were dispensed on the nonwoven fabric-type scaffold sample prepared in Examples 1 to 3. Samples were cut using a 5 mm diameter punch and placed on a 96-well plate, and 2 x 10 ⁴ cells were seeded onto the scaffold and dispersed evenly therein. In order to determine the initial number of cells, the same number of cells were seeded in an empty well, allowing the cell attachment rate to be calculated.

At 4 hours after cell seeding, the cell-seeded scaffold sample was transferred to a new well and MTT ((3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay , The absorbance of the cells adhering to the sample was compared with the absorbance of cells seeded at the early stage, and the cell adhesion rate was calculated as follows. The results are shown in Fig.

Cell Adhesion (%) = [As / Ac] x 100

As: absorbance of cells attached to scaffold

Ac: Absorbance of cells initially seeded in scaffold

Referring to FIG. 9, the degree of cell growth by PLGA / ES nonwoven fabric content was examined. As the content of PLGA was increased, cell adhesion was improved.

Experimental Example 4: Cell growth assay

NIH 3T3 (mouse embryonic fibroblast cell line) was prepared as a cell, and the cells were dispensed on the nonwoven fabric-type scaffold sample prepared in Examples 1 to 3. Samples were cut using a 5 mm diameter punch, placed in 96-well plates, and seeded so that 2 x 10 ⁴ cells could be spread evenly over the scaffold.

After 24 h of cell seeding, all samples were transferred to 24-well plates and then MTS ([3- (4,5-dimethylthiazol-2-yl) -5- (3 The number of cells grown through the MTS assay was quantitated and the results are shown in Figure 10. The number of cells known before the MTS treatment was measured By drawing the standard curve to show the relationship between the absorbance and cell number, the absorbance value obtained from the actual scaffold was assigned to the standard curve and the exact cell number could be quantified.

Referring to FIG. 10, the degree of cell growth was checked by PLGA / ES nonwoven fabric content, and it was confirmed that the higher the content of PLGA, the better the cell growth.

Example 4 (Production of cylindrical scaffold )

The PET scrap was prepared by using a 12Gauge 20needle circular knitting machine, PLA warp knitting yarn (75de / 36fila) was inserted into the knitted yarn, and the twist yarn was pulled up to 15-20% of the conventional length to make a cylindrical scaffold. The manufacturing process is schematically shown in Fig.

Example 5 (Production of cylindrical scaffold )

A cylindrical scaffold was prepared in the same manner as in Example 4, except that PLA warp yarns and PLGA warp yarns (220de / 112fila) were used at a weight ratio of 25/75 instead of PLA warp yarns (75de / 36fila).

Example 6 (Production of cylinder type scaffold )

A cylindrical scaffold was prepared in the same manner as in Example 4, except that PLGA false twist yarn (220de / 112fila) was used instead of PLA false twist yarn (75de / 36fila).

Experimental Example 5: Analysis of biodegradation rate according to the amount of irradiation

The cylindrical scaffolds prepared in Examples 4 to 6 were cut at intervals of 1 cm and 50 ml of a phosphate buffered saline (PBS, pH 7.4) solution was added to a 50 ml Falcone tube. The samples were immersed in a 50 ml Falcon tube, rpm), and the pH of the PBS was measured at each time point to confirm the biodegradation rate. The evaluation results for Examples 4 to 6 are shown in Figs. 12 to 14, respectively.

On the 12th day and the 33th day after incubation, the sample was taken out and completely dried in a convection oven at 40 ° C. After that, the sample was frozen in liquid nitrogen to cut a cross section (cut in half in the longitudinal direction) (FE-SEM, SU 8010) to examine the cross-section and the surface. SEM observation results of the gamma ray irradiation amounts OkGy, 30 kGy, and 50 kGy in Examples 4 to 6 are shown in Figs. 15 to 17, respectively.

Comparative Example 1 (Production of nonwoven fabric type scaffold)

Nonwoven fabric type scaffold was prepared in the same manner as in Example 1 except that PLGA fiber (fineness: 2 denier, fiber length: 3 mm, melting point: 210 ° C) was used alone.

Experimental Example 6: Analysis of cell growth according to gamma irradiation

Each sample prepared in Example 3 and Comparative Example 1 was irradiated at different doses at 0, 10, 20, 30, and 50 kGy using a gamma irradiation facility (MDS Nordion Inc C-188). NIH 3T3 (mouse embryonic fibroblast cell line) was prepared as a cell, and cells were dispensed on the nonwoven fabric-type scaffold sample prepared according to the irradiation amount.

Samples were cut into 1 cm x 1 cm, placed in 48 well plates, and seeded so that 5 x 10 ⁴ cells could be distributed evenly over the scaffold and inside. After 24 h of cell seeding, all samples were transferred to 24 well plates. MTS ([3- (4,5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl) The number of cells grown through the MTS assay was determined and the results are shown in Figure 18. The number of cells known at the time of MTS treatment was measured at various dilution ratios By drawing the standard curve to show the relationship between the absorbance and the cell number, the absorbance value obtained from the actual scaffold was assigned to the standard curve and the exact cell number could be quantified.

18, in comparison with the scaffold of Comparative Example 1 in which the scaffold of Example 3 made of wet nonwoven fabric by combining PLGA fiber and low melting point ES fiber, which is a biodegradable material, was a wet nonwoven fabric formed only of PLGA fibers, It was confirmed that the degree of cell growth was improved over all the gamma irradiation dose.

In Example 3, the PLGA fiber is biodegraded to provide a space for cell growth while the cell growth is being performed, the ES fiber is not biodegraded, and the PLGA fiber is biodegraded and the scaffold skeleton is maintained so that the provided cell growth space is not collapsed . On the other hand, in Comparative Example 1, which is a non-woven fabric type spade made of PLGA fibers, which is a biodegradable material, the space of the scaffold can be secured by the biodegradation of the PLGA fiber, but there is no skeleton to support such space with time Collapse and cell growth becomes insufficient. As a result, in the nonwoven fabric-type scaffold of Example 3, since the space formed by the biodegradation of the PLGA fibers plays a role of supporting the ES fibers, it is possible to confirm the growth of many cells by providing the optimal conditions for cell growth .

Claims

A three-dimensional fiber-type scaffold having a plurality of pores,

Wherein the scaffold includes a fiber structure having two kinds of fibers having different biodegradation rates,

Wherein the two types of fibers having different biodegradation rates include a biodegradable first fiber and a biodegradable second fiber having a slower biodegradation rate than the first fiber or a biodegradable first fiber and a non biodegradable second fiber Dimensional fibrous scaffold.
The method according to claim 1,

Wherein the biodegradable first fiber has a higher melting point or no melting point than the biodegradable second fiber or the non-biodegradable second fiber.
The method according to claim 1,

Wherein the average size of the pores of the scaffold increases with time.
The method according to claim 1,

Wherein the biodegradable first fibers and the biodegradable second fibers are selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid, polyhydroxybutyric acid- A three-dimensional fibrous scaffold comprising a copolymer of valeric acid, collagen, hyaluronic acid, cellulose oxide, chitosan, chitin, gelatin, silk fibroin or a mixture of two or more thereof.
The method according to claim 1,

Wherein the non-biodegradable second fiber comprises a polyolefin, a polyester, a polyamide, or a mixture of two or more thereof.
The method according to claim 1,

Wherein the biodegradable first fiber comprises polyglycolic acid (PGA), a copolymer of polylactic acid-glycolic acid (PLGA), or a mixture of two or more thereof, the biodegradable second fiber comprises polylactic acid,

Or the biodegradable first fiber is selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, copolymers of polylactic acid-glycolic acid, polyhydroxybutyric acid, polyhydroxyvaleric acid, polyhydroxybutyric acid-valeric acid copolymer, Wherein the non-biodegradable second fiber comprises at least one of polypropylene, polyethylene, polyethylene terephthalate (PET), polyethylene terephthalate (PET), polyethylene terephthalate (PTT), polycyclohexanedimethanol terephthalate (PCT), and polyethylene naphthalate (PEN), nylon 6, nylon 6,6, nylon 4, and nylon (polytetramethylene terephthalate 4,6 or a mixture of two or more thereof.
The method according to claim 1,

Said scaffold being in the form of a nonwoven; Fabric type; Knitted form; Fiber bundle type; A cylindrical shape having a fiber bundle and a tube into which the fiber bundle is inserted, or a mixture of two or more thereof.
The method according to claim 1,

Wherein the biodegradation rate can be controlled by gamma irradiation.
The method according to claim 1,

Wherein the fibrous structure further comprises one or more fibers having different biodegradation rates.