WO2018105980A1 - Fiber-filled three-dimensional support and manufacturing method therefor - Google Patents

Abstract

The present invention relates to a fiber-filled three-dimensional support and a manufacturing method therefor. The fiber-filled three-dimensional support of the present invention is advantageous for use as a tissue culture support for cell culture, proliferation, and differentiation, since a 3D printing structure formed of an in vivo absorbent polymer material is used as a frame and microfilaments formed of in vivo absorbent polymer material are filled in the three-dimensional direction inside the structure to achieve complexation, so that a pore size is properly controlled depending on a use of the support, a space sufficient for cell proliferation is provided, and an intercellular space interconnection is improved, thereby allowing three-dimensional cell proliferation.

Description

Fiber-filled three-dimensional support and manufacturing method thereof

The present invention relates to a fiber-filled three-dimensional support and a method for manufacturing the same, and more specifically, to a 3D printing structure made of an in vivo absorbent polymer material, and filling the inside of the structure with microfilament yarns made of an in vivo absorbent polymer material. By combining, providing a space sufficient for cell proliferation and at the same time provide a pore size suitable for cell support to improve intercellular space interconnection (interconnection), three-dimensional fiber-filled three-dimensional support and its manufacturing method It is about.

Tissue engineering refers to a technology that attempts to maintain, improve, and restore the function of a living body by making a substitute for living tissue based on basic concepts and techniques of life science, medicine, and engineering and implanting it into a living body. will be. The actual implementation of biotissue engineering involves collecting the necessary tissue from the patient's body, separating the cells from the tissue pieces, and proliferating the separated cells by the necessary amount through cultivation, and incubating the cells in a biodegradable polymer support having a porosity for a certain period of time. The formed scaffolds (also called 'cell culture supports') are implanted back into the human body.

Thus, a tissue engineering support used for regeneration of human tissue refers to a biocompatible material that is combined with a transplantation cell and implanted in a disease and injury site of the human body to effectively serve as a tissue regeneration and regeneration helper for cells.

Such a support should basically have good adhesion of tissue cells and have mechanical strength so that the tissue cells adhered to the material surface can form a tissue having a three-dimensional structure. It should also serve as an intermediate barrier between the transplanted and host cells, which requires nontoxic biocompatibility that does not result in coagulation or inflammatory reactions after transplantation.

Conventional techniques for producing three-dimensional supports include salt-casting particulate leaching, gas foaming, fiber meshes / fiber bonding, phase separation, Melt molding, freeze drying, electrospinning and the like have been tried.

Among them, the support fabricated by the fiber mesh / fiber bonding method or the fiber mesh method has the advantages of a simple process and high porosity, but there is a problem in a large amount of cell proliferation due to poor structural stability and difficulty in securing sufficient space, and produced by the solvent casting method. The support has a high porosity, but it is difficult to control the pore shape and there is a risk of remaining organic solvent.

In addition, the nanofiber support prepared by the electrospinning method has an excellent advantage in cell adhesion but is limited to a two-dimensional structure because the cells are not proliferated in three dimensions due to pores smaller than the cell size, in particular, there is a problem of the remaining organic solvent.

In other words, the conventional tissue engineering support has a limited three-dimensional shape and cannot precisely control the pore size, porosity, and inter-pore interconnectivity according to the designer's intention. There is a problem that high, reproducibility is significantly reduced.

In addition, there is a problem that the shape and the implantation site does not exactly match when implanted in a specific position.

In this regard, the Republic of Korea Patent No. 1506704 recently realized the shape of the intended scaffold by molding into a three-dimensional shape by 3D printing, and provides a porous scaffold to freely control the size and shape of the pores, Patent No. 2016-95481 also provides a support for filling bone defects with excellent bone regeneration effect by using three-dimensional printing, and it is economical because no expensive mold is required for molding, and also supports various types of support It is suggested that it can be done.

Furthermore, Korean Patent No. 1371671 discloses a scaffold having complex pores, which can further increase porosity by allowing pores to be formed in the strand itself forming the scaffold, thereby further improving adhesion rate of tissue cells.

However, the support by the 3D printing has the advantage that it can be manufactured by tailoring the structural stability and damage site, but difficult to implement a micro-microstructure, the ability to support and maintain a cell having a size of tens to hundreds of micrometers in the support is inferior There is.

Accordingly, the present inventors have made efforts to improve the problems of the conventional tissue engineering support, as a skeleton of the three-dimensional structure obtained by 3D printing that is structurally stable and can be customized to the damage site, the micro-thick fibers inside the structure The present invention was completed by verifying a function of stably supporting, containing, and maintaining cells by filling and controlling the size of the pores, the porosity, and the inter-pore interconnectivity according to the designer's intention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a fibrous-filled three-dimensional support in which a 3D printing structure made of an in vivo absorbent polymer material is used as a skeleton and is filled with microfilament yarns in order to form a pore size suitable for the use of the support. .

Another object of the present invention is to provide a method for preparing the fiber-filled three-dimensional support.

The present invention provides a fiber-filled three-dimensional support having excellent intercellular space connectivity by filling microfilament yarns made of in vivo absorbent polymer material in a three-dimensional direction in a 3D printing structure made of in vivo absorbent polymer material.

The 3D printing structure is a three-dimensional lattice (lattice) structure, more preferably in the form of a multi-layer net, the net line thickness is 200 to 1,000㎛, the net spacing can be controlled to 200 to 1,000㎛, In the form of the multilayer net, the interlayer height is made from 200 to 1,000 mu m.

In addition, the filament yarn is a multi-filament of at least 10 or more melt-spun at a Tm or more of the material, wherein, the fiber diameter of the filament yarn is preferably 5 to 30㎛.

Furthermore, the present invention is a first step of manufacturing a 3D printing structure of the porous three-dimensional structure customized to the damaged site by 3D printing using the in vivo absorbent polymer material, to melt-spun the absorbent polymer material in vivo to form a microfilament yarn It provides a method for producing a three-dimensional fiber-filled support comprising a second step, the third step of filling the microfilament yarn inside the 3D printing structure to control the filling density and the fourth step of heat setting after the filling.

In the third process, when the microfilament yarn is filled by compounding by hooking a needle hook and passing through the interior space of the 3D printing structure, or fabricated by 3D printing, each layer is formed by 3D printing. At the same time, the microfilament yarns are arranged in a layered manner and simultaneously composited in an in-situ.

In addition, the fourth step is to perform heat setting at a temperature in the range of Tg (glass transition temperature) and Tm (melting temperature) of the in vivo absorbent polymer material.

The fiber-filled three-dimensional support according to the present invention has a 3D printing structure made of an in vivo absorbent polymer material and a microfilament yarn is filled in the structure, so that a suitable pore size can be controlled according to the use of the support.

The 3D printing structure ensures sufficient space to secure the number of cells and fills the microfilament yarns in the three-dimensional direction inside the structure to control the size of the pores, control the porosity and the filling density, thereby increasing reproducibility. In particular, cell proliferation is possible in three dimensions by enhancing interconnection between cells.

The fiber-filled three-dimensional scaffold of the present invention improves the function of supporting, containing, and maintaining the cells, and effectively supports the damaged area by tailoring the damaged area.

1 is a 3D printing structure produced in Example 1 of the present invention, (a) is a horizontal × vertical shape, (b) is a height shape,

Figure 2 shows the (a) front, (b) top and (c) side of the 3D printing structure of Figure 1 cut to a size of 4mm × 4mm × 7.2mm,

3 is a fibrous filled solid support prepared by filling two layers of microfilament yarns in a 3D printing structure in Example 1 of the present invention, (a) is an upper portion, and (b) is an enlarged image of 19 times the side surface Micrograph,

Figure 4 is a three-dimensional fibrous filled support of Figure 3, (a) is the top, (b) is an image micrograph magnified 160 times the side,

5 is a fibrous-filled three-dimensional support fabricated by filling microfilament yarns in four layers in a 3D printing structure in Example 2 of the present invention, (a) is a photo before cutting and (b) after cutting,

6 is a schematic view of the front side and a part of the enlarged photograph of the fiber-filled three-dimensional support produced in Example 3 of the present invention,

Figure 7 is a schematic diagram of a cross-sectional view of the fiber-filled three-dimensional support of Figure 6,

8 is a photograph evaluating the fiber diameter of the microfilament yarn inserted into the fiber-filled three-dimensional support of FIG.

FIG. 9 is a photograph evaluating the pore size of the microfilament yarn inserted into the fiber-filled three-dimensional support of FIG. 6,

FIG. 10 is an image result of cell proliferation in microfilament yarns internally filled in the fiber-filled three-dimensional support of FIG. 6,

11 is a process flow diagram of a method for producing a fibrous packed solid support of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a fiber-filled three-dimensional support having excellent intercellular space connectivity by filling microfilament yarns made of in vivo absorbent polymer material in a three-dimensional direction in a 3D printing structure made of in vivo absorbent polymer material.

1) 3D printing structure of porous 3D structure

In the fiber-filled three-dimensional support of the present invention, the 3D printing structure is a porous three-dimensional structure with excellent structural stability and can be formed in a custom shape, and formed into a three-dimensional lattice structure using a selected bioabsorbable polymer material. do. More preferably, it may include a multilayer net form, honeycomb form, and the like.

1 is a 3D printing structure of 24mm × 24mm × 7.2mm (width × length × height) size manufactured in Example 1 of the present invention, and FIG. 2 shows the porous 3D structure having a size of 4mm × 4mm × 7.2mm. (A) front side, (b) upper side, and (c) side which were cut are shown.

The 3D printing structure can identify a three-dimensional lattice structure in which two layers exist in a side surface and two layers in independent spaces exist in a neighboring side.

In the case of applying a tubular circular piece as a conventional support, the shape is fixed in a cylinder type, while the 3D printing structure of the present invention can be produced in a three-dimensional lattice shape of a desired shape by 3D printing, for example, a bone-shaped multilayer net shape. Since it can be produced in the three-dimensional lattice structure of the can be designed in a customized structure of the damaged area.

3 is a three-dimensional fibrous support filled with a microfilament yarn inside the 3D printing structure in Example 1 of the present invention (a) is the top, (b) is an image microscope magnified 19 times the side It is a photograph, and FIG. 4 is a 160-time magnification (a) of the upper part, (b) is a side image micrograph result. From the above results, the microfilament yarns are smoothly filled inside the 3D printing structure, and the size of the internal voids can be adjusted by changing the number of strands of the microfilament yarns to be inserted.

In addition, Figure 5 is a fiber-filled three-dimensional support prepared by filling the microfilament yarn in four layers in the 3D printing structure in Example 2 of the present invention (a) is a photo before cutting, (b) after cutting.

The fiber-filled three-dimensional scaffold in which the microfilament yarns are inserted in four directions from the above can be applied by cutting the edges to a size convenient for cultivation.

In addition, Figure 6 is a schematic and partially enlarged actual image photograph of the front of the fiber-filled three-dimensional support produced in Example 3 of the present invention, Figure 7 is a cross-sectional view of the fiber-filled three-dimensional support observed from the side It is a schematic diagram.

At this time, the 3D printing structure is produced in the form of a multi-layer net, preferably the net line thickness is 200 to 1000㎛, more preferably 200 to 400㎛, net spacing 200 to 1000㎛, more preferably 200 To 800 μm structure is designed. Therefore, the left and right and upper and lower pores of the formed mesh is preferably at least 200㎛.

In addition, from the cross-sectional photograph of the 3D printing structure observed from the side, the height between the layers of the multi-layered net structure is 200 to 1000 µm, more preferably 200 to 800 µm.

The material of the 3D printing structure of the present invention is not particularly limited as long as it is an absorbent polymer material in vivo, and a preferred example thereof is polyglycolic acid, polylactic acid (D, L, DL), polycaprolactone, glycolic acid-lactic acid (D, At least one of L, DL) copolymer, glycolic acid-ε-caprolactone copolymer, lactic acid (D, L, DL) -ε-caprolactone copolymer, and poly (p-dioxanone) synthetic polymer material is used. do.

The 3D printing structure made of the above material has a sufficient pore to form a cell supporting, including, and maintaining a function well, and can control pore size and porosity, thereby increasing reproducibility. Thus, the 3D printing structure inserts and fills fibers of several tens of micrometers in thickness to provide an environment suitable for cell size.

2) micro filament yarn

The fiber-filled three-dimensional support of the present invention has a 3D printing structure as a skeleton, but the 3D printing structure is complex by filling and fixing the microfilament yarn inside the structure, which makes it difficult to implement the micro-microstructure, the size of tens to hundreds of micrometers Designed to support, contain and maintain cells reliably.

The material of the filament yarn is not particularly limited as long as it is an in vivo absorbent polymer material, and preferred examples thereof include polyglycolic acid, polylactic acid (D, L, DL), polycaprolactone, glycolic acid-lactic acid (D, L, DL). At least one of a copolymer, a glycolic acid-ε-caprolactone copolymer, a lactic acid (D, L, DL) -ε-caprolactone copolymer, and a poly (p-dioxanone) synthetic polymer material is used.

In addition, the microfilament yarn is more preferably 10 or more multifilament melt-spun at a Tm or more of the material, the micro multifilament may be used as a (Fraw Textured Yarn) or twisted fibers (Crimp Yarn).

8 is a photograph of the fiber diameter of the microfilament yarns of the fiber-filled three-dimensional support of the present invention. Preferably, the fiber diameter of the microfilament yarns is 5 to 30 µm.

Particularly, if the fiber diameter is less than 5 μm or micronized, the inter-fiber pores are narrowed and intercellular space interconnection is poor, which makes it difficult to proliferate cells in three dimensions. The fiber density is reduced, the cell support, containment, retention performance is reduced, the stiffness is increased, there is a disadvantage in that workability and ease of operation.

9 is a photograph evaluating the pore size of the microfilament yarn of the fiber-filled three-dimensional support of the present invention, the preferred pore size has a pore of 1 ~ 150㎛, more preferably 5 ~ 100㎛. At this time, if the pore size is less than 1 μm, the pores between fibers are small, making cell proliferation difficult during cell culture, and the content of cells or drugs that can be delivered in vivo is low, and the utility as a support that can be used for medical purposes is inferior. In addition, when the pore size exceeds 150 µm, the voids between the fibers become too large and the retention capacity of cells or drugs tends to be low, which is not preferable.

10 is a result of cell proliferation in microfilament yarns filled internally in the fibrous-filled three-dimensional scaffold of the present invention. As a result of evaluating cell proliferation performance 4 days after culturing the cells in the microfilament, the pore size of the microfilament cells It can be confirmed that it is preferable for proliferation.

The fiber-filled three-dimensional support of the present invention is a structure in which a micro multifilament imparted with bulky properties is inserted into a 3D printing structure, and cells can grow well in many pores of a bulky micro multifilament internal space, and micro multi according to size Since the number of filaments can be adjusted, the structure can be applied to cells of various sizes. In addition, the size of the pores can be changed in various ways, which is a very suitable support for cell growth.

Furthermore, the present invention is a process flow diagram of the manufacturing method of the fiber-filled three-dimensional support of the present invention shown in Figure 11, by using a 3D printing in vivo absorbent polymer material to customize the 3D printing structure of the porous three-dimensional structure customized to the damage site Manufacturing first process,

A second step of forming a microfilament yarn by melt spinning the absorbent polymer material in vivo;

A third process of filling the microfilament yarns into the 3D printing structure;

It provides a method for producing a fibrous filler solid support consisting of a fourth step of heat setting after the filling.

In the manufacturing method of the present invention, the first step is a process of forming a 3D printing structure from the absorbent polymer material, and the net line, the gap and the interlayer height are manufactured to the desired pore size by tailoring the damage site by the preset 3D printing.

In the manufacturing method of the present invention, the second step is a step of forming a microfilament yarn using the absorbent polymer material in vivo, and after spinning into monofilament or multifilament yarn by a melt spinning method, more preferably 10 or more Plywood twist can be used to use biodegradable multifilament twisted yarn or crimp yarn.

The biodegradable multifilament twisted yarn is passed through a yarn of monofilament and multifilament having a thickness of 50 to 500 deniers through a combustor such as a roller type combustor or a disk type combustor and twisted in the S direction to the Z direction to give swelling sound. To give.

At this time, the fiber diameter of the microfilament yarn is 5 to 30㎛, satisfying the physical properties of the strength of 2.0 ~ 9.0 g / d and 20 to 80% elongation, it is possible to minimize the generation of trimming and deterioration in the subsequent draw twist process.

In the manufacturing method of the present invention, the third step is to fabricate the 3D printing structure in the first step and the microfilament yarn in the second step, respectively, and then hook the microfilament yarn to the needle hook It can be filled and compounded by passing the space inside the 3D printing structure.

Alternatively, when the 3D printing structure is manufactured, the microfilament yarns may be simultaneously layered by in-situ by laminating each layer by 3D printing and simultaneously layering the microfilament yarns.

In the manufacturing method of the present invention, the fourth step is a step of heat-setting after filling and complexing in the third step, it is completed by performing at a temperature of Tg (glass transition temperature) and Tm (melting temperature) range of the absorbent polymer material in vivo do.

Thereafter, the obtained support may be further washed with water to sterilize.

<Example 1>

1. Manufacturing process of 3D printed structure of porous 3D structure

A polycaprolactone (Poly-Caprolactone) polymer chip, a biodegradable polymer having a number average molecular weight (Mw) of 50,000, was prepared by 3D printing under the output conditions shown in Table 1 below. In this case, polycaprolactone is a material near the melting point 60 and is suitable for application to the extrusion lamination method.

1 is a 3D printing structure designed in the present invention, (a) is a horizontal × vertical shape, (b) is a height shape as a total of 24mm × 24mm × 7.2mm (width × height × height) 3D printing Figure 2 shows a structure having (a) the front (b) top and (c) side cut the structure of Figure 1 to a size of 4mm x 4mm x 7.2mm.

Referring to the structure of FIG. 2, there are two layers on the side and two layers of independent space on the neighboring side.

2. Micro filament yarn manufacturing process

Polylactic acid (PLLA) polymer chips were spun into 75 denier / 36 filament multifilament yarns by melt spinning. Using a roller-type combustor, PLLA twisted yarn (DTY) having a twist in the Z direction was manufactured.

3. Filling and heat setting process

Twenty PLLA twisted yarns made of the microfilament yarns were inserted and filled into the inner spaces (two layers) of the 3D printing structure using circular needle hooks, respectively, and both sides of the twisted yarns were stretched by 15%. The tension gave PLLA false-twist yarns to form pores within the 3D printing structure and impart bulkiness. After heat-setting at 50 ℃ to produce a fiber-filled three-dimensional support.

The fabric filled solid support was washed with water and sterilized.

<Example 2>

In the filling and heat setting process of Example 1, 20 PLLA twisted yarns were filled in the inner space (two layers) of the 3D printing structure and filled, and 20 PLLA twisted yarns were also applied to the neighboring side surfaces in the same manner. Insert was carried out in the same manner as in Example 1 except that the four layers were filled and then tensioned.

<Example 3>

1. Fabrication process of 3D printed structure of porous 3D structure

Using a polycaprolactone polymer chip, a multi-layered net-shaped 3D printed structure having a thickness of 200 μm, a net spacing of 800 μm, and an interlayer height of 800 μm was produced by 3D printing.

2. Micro filament yarn manufacturing process

Polylactic Acid-Glycolic Acid Copolymer (PLGA) Synthetic Polymer Chips of Lactide and Glycolide Copolymerized in a Weight Ratio of 10:90 were spun into PLGA (10:90) 110de / 56fila multifilament yarn by melt spinning method. PLGA combustible yarn (DTY) having twist in the Z direction was manufactured using a roller combustor. In this case, the average fiber diameter was 13.8 µm and the pore size was 47.6 µm.

3. Filling and heat setting process

The micro multifilament yarn was filled in a manner of hooking a needle hook and passing through the interior space of the 3D printing structure, and heat-fixed at 50 ° C. to prepare a fibrous filler solid support. Then, washed with water and sterilized.

<Example 4>

When manufacturing the 3D printing structure by 3D printing, one layer (Layer) by laminating each layer (Layer) by 3D printing of the DTY twisted yarn, which is a composite of the micro multifilament yarn manufactured in Example 1 in-situ ) was completed by co-complexing, and heat-set at 50 ° C to prepare a porous three-dimensional support.

Experimental Example 1

As a result of photographing the porous three-dimensional support prepared in Example 1 by using an image microscope, Figure 3 is a three-fold enlarged fiber-supported three-dimensional support filled with microfilament yarn inside the 3D printing structure (a) is the top, ( b) is a side view, and FIG. 4 is an image of (a) the upper side and (b) the side view of the fiber-filled three-dimensional support 160 times larger.

As a result, it was confirmed that the PLLA twisted yarn was well-filled inside the 3D printing structure, and thus the number of strands of the inserted PLLA twisted yarn was changed to

Experimental Example 2

The porous three-dimensional support prepared in Example 2 was photographed using an image microscope, and FIG. 5 is a cut of the fiber-filled three-dimensional support in which microfilament yarns are inserted into four layers in the 3D printing structure of the present invention. Before, and (b) is a photograph after cutting.

The fiber-filled three-dimensional scaffold in which the PLLA twisted yarns are inserted in four directions from the above can be used by cutting the edges to a size convenient for incubation in a 48-well microplate culture dish.

Experimental Example 3

As a result of photographing the porous three-dimensional support prepared in Example 3 using an image microscope, it was confirmed that the filament yarn was densely and smoothly filled into the 3D printing structure formed by 3D printing as a skeleton. At this time, the average pore size was 47 ㎛ to provide a space that can be stably proliferated cells having a size of several tens to hundreds of micrometers.

Experimental Example 4

Incubated 2 × 10 ⁴ cells / well (NIH3T3 fibroblasts cell) in the micro multifilament yarn prepared in Example 3 and after 4 days by using an electron microscope confirmed that the cells proliferate in the three-dimensional direction.

As described above, the present invention provides a fiber-filled three-dimensional support in which a 3D printing structure made of an in vivo absorbent polymer material is a skeleton and a microfilament yarn is filled in the structure in a three-dimensional direction.

The fiber-filled three-dimensional scaffold of the present invention has a sufficient space to secure the number of cells due to the 3D printing structure, and the cell support and intercellular space connectivity (interconnection) are improved due to the filling of the microfilament yarn inside the structure in three dimensions. Cell proliferation is possible.

Accordingly, the fiber-filled three-dimensional scaffold of the present invention improves the function of supporting, containing, and maintaining cells, and has excellent connectivity between intercellular spaces, which is advantageous for cell culture, delivery, or drug delivery on a three-dimensional structure.

While the invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the invention, and such modifications and variations belong to the appended claims.

Claims (8)

1. Inside the 3D printing structure made of in vivo absorbent polymer material,

   A fiber-filled three-dimensional scaffold having excellent cell support and intercellular space connectivity by filling microfilament yarns made of in vivo absorbent polymer material in three dimensions.
2. The three-dimensional printing structure of claim 1, wherein the 3D printing structure is in the form of a multi-layer net, the net line thickness is 200 to 1000㎛, and the net spacing is 200 to 1000㎛.
3. The fiber-filled three-dimensional support according to claim 2, wherein the multilayer net form has an interlayer height of 200 to 1000 µm.
4. The fiber-filled three-dimensional support according to claim 1, wherein the filament yarn is a multi-filament of at least 10 polymers melt-spun at a Tm or more of the material.
5. The fibrous filler support according to claim 1, wherein the filament yarn is a microfilament yarn having a fiber diameter of 5 to 30 µm.
6. A first process of manufacturing a 3D printing structure having a porous three-dimensional structure customized for damage by 3D printing using an in vivo absorbent polymer material,

   A second step of forming a microfilament yarn by melt spinning the absorbent polymer material in vivo;

   A third process of filling the microfilament yarns into the 3D printing structure;

   Method of producing a three-dimensional fiber-filled support comprising a fourth step of heat setting after the filling.
7. The microfilament yarn of claim 6, wherein the third process fills the microfilament yarn by hooking the needle hooks and passes through the interior space of the 3D printing structure, or at the same time laminating each layer by 3D printing when the 3D printing structure is manufactured. Method of producing a three-dimensional fiber-filled scaffold support, characterized in that the layered layer is filled in one step ( in-situ ).
8. The method of claim 6, wherein the fourth step is heat-fixed at a temperature in the range of Tg (glass transition temperature) and Tm (melting temperature) of the in vivo absorbent polymer material.

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