KR101686788B1 - 3-dimensional scaffold having patterns and manufacturing method thereof - Google Patents
3-dimensional scaffold having patterns and manufacturing method thereof Download PDFInfo
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- KR101686788B1 KR101686788B1 KR1020150097603A KR20150097603A KR101686788B1 KR 101686788 B1 KR101686788 B1 KR 101686788B1 KR 1020150097603 A KR1020150097603 A KR 1020150097603A KR 20150097603 A KR20150097603 A KR 20150097603A KR 101686788 B1 KR101686788 B1 KR 101686788B1
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- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
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Abstract
Description
BACKGROUND OF THE
Recently, nanofiber has been actively studied as a functional material. Here, the nanofiber refers to a fiber having a particle diameter of less than 1 占 퐉. Such nanofibers are known to exhibit high cell affinity as well as being used in various industrial fields such as batteries, high-efficiency nanofiber filters, lightweight functional coatings, cell carriers for tissue regeneration, biosensors, and drug delivery systems have.
Therefore, although such nanofibers are used in scaffolds, there are difficulties in realizing the three-dimensional structure besides the two-dimensional arrangement, and the physical properties of the nanofibers and the complexity of the muscle tissue structure.
In addition, the scaffold is an artificially created ECM for tissue establishment and cell function control, and the extracellular substance is an extracellular matrix (ECM), which is composed mainly of organic polymers such as proteins and polysaccharides Solid matter is present and serves as structural support for tissue and as a cell adhesion promoter. When cells attach to ECM, intracellular signal transduction is activated and basic cell functions such as cell morphology, proliferation, and cell death are controlled.
Since ECM exists as a crosslinked gel state or a porous network in living tissue, much attention has been focused on gel-like scaffolds and porous scaffolds similar to bio-ECMs in terms of structure in design of scaffolds.
However, conventional gel-type scaffolds and porous scaffolds have limitations in realizing a scaffold for regenerating tissue having a composite hierarchical structure of nano and micro-scale, such as muscle tissue.
Therefore, in order to solve such problems, a three-dimensional scaffold which can form muscle tissue very similar to the actual one by differentiating the muscle cells into a hierarchical structure with a nano / micro composite scale while maintaining excellent physical properties of the nanofiber, Development of the manufacturing method is required.
The object of the present invention is to provide an excellent cell adhesion and cell induction function with a high surface area by constituting a three-dimensional scaffold with nano-fibers electrospun as an optimal biocompatible material.
Further, by forming a pattern on the scaffold by a femtosecond laser with an optimal wavelength, frequency, pulse energy and ablation rate, it is possible to effectively realize a pattern in the nanofiber, and there are few defects such as surface damage and cracks Dimensional scaffold suitable for cell differentiation because the surface of the pattern is smooth.
It also aims to form a hierarchical structure of nano / micro complex scales similar to muscle tissue as well as causing active differentiation of the source cells by optimizing the pattern shape, width and spacing between the patterns.
According to another aspect of the present invention, there is provided a patterned three-dimensional scaffold comprising a plurality of patterns arranged in one direction on a three-dimensional scaffold, and the scaffold is formed by electrospun nano- And the pattern may be in the form of a groove.
The distance between the patterns may be on average from 5 to 100 占 퐉, the distance between the patterns may be on average from 10 to 25 占 퐉, and the width of the pattern may be on average from 3 to 10 占 퐉.
Also, the patterns may be arranged parallel to each other, and the pattern may be formed by a femtosecond laser, and the femtosecond laser forms the pattern at an ablation rate of 10 to 60 mm / s at a frequency of 5 to 15 kHz can do.
The electrospun nanofiber may be selected from the group consisting of aliphatic polyesters, polyalkylene oxides, polydimethylsiloxanes, polycaprolactones, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycan, And a polymer selected from a combination thereof.
The aliphatic polyester may be selected from the group consisting of lactic acid (D- or L-), lactide, poly (lactic acid), poly (lactide) glycolic acid, poly (glycolic acid), poly (glycolide), glycolide, Co-glycolide), poly (lactic acid-co-glycolic acid), and combinations thereof.
Next, a manufacturing method of a three-dimensional scaffold in which a pattern of the present invention is formed includes a scaffold forming step of forming a three-dimensional scaffold made of nanofibers by an electrospinning method; And a pattern forming step of forming a pattern by irradiating a femtosecond laser onto the three-dimensional scaffold.
The scaffold-forming step may form the chamber-shaped three-dimensional scaffold by collecting the polymer solution into the chamber by radiating the high-voltage solution through a nozzle.
The polymer solution may be at least one selected from the group consisting of polyalkylene oxide, polydimethylsiloxane, polycaprolactone, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides, aliphatic polyesters, Wherein the aliphatic polyester is selected from the group consisting of lactic acid (D- or L-), lactide, poly (lactic acid), poly (lactide) glycolic acid, poly (glycolic acid), poly (glycolide), glycolide, Poly (lactide-co-glycolide), poly (lactic-co-glycolic acid), and combinations thereof.
Wherein the pattern formation step comprises moving the femtosecond laser having a wavelength within a range of 400 to 800 nm in parallel in one direction and irradiating a laser beam having a pulse energy of 0.5 to 6 μJ using a 5 to 10 magnification lens at a frequency of 5 to 15 kHz at a frequency of 10 to 60 mm / dimensional scaffold at an ablation rate of s to form the pattern. In the pattern formation step, a plurality of the patterns may be formed in a groove shape, and may be parallel to each other.
In addition, the distance between the patterns may be an average of 10 to 25 占 퐉, and the width of the pattern may be an average of 3 to 10 占 퐉.
The pattern can control the adhesion and differentiation of myoblasts. On the three-dimensional scaffold after the pattern formation step, the myoblasts are differentiated to form myotubes .
In addition, the mean arrangement angle of the myotubes may be 1 to 15 degrees, and the myotube may be hierarchically formed along the nanofibers and the pattern to realize muscle tissue.
INDUSTRIAL APPLICABILITY According to the present invention, by forming a three-dimensional scaffold with nanofibers electrospun as a biocompatible material, excellent cell adhesion and cell-inducing functions can be provided with a high surface area.
Further, by forming a pattern on the scaffold by a femtosecond laser with an optimal wavelength, frequency, pulse energy and ablation rate, it is possible to effectively realize a pattern in the nanofiber, and there are few defects such as surface damage and cracks , The surface of the pattern is smooth, and a three-dimensional scaffold suitable for cell differentiation can be provided.
In addition, by optimizing the pattern shape, width, and spacing between the patterns, it is possible not only to cause active differentiation of the source cells, but also to form a hierarchical structure of nano / micro complex scales similar to muscle tissue.
Fig. 1 is a schematic view of a method of manufacturing a three-dimensional scaffold in which a pattern of the present invention is formed
Fig. 2 is a flowchart sequentially showing a method of producing a patterned three-dimensional scaffold
Fig. 3 is a schematic view simulating a scaffold formation step (S10) of a method of manufacturing a three-dimensional scaffold in which a pattern is formed
FIG. 4 is a comparative chart comparing the irradiation effect of the femtosecond laser with other lasers in the pattern forming step (S20) of the method of manufacturing a three-dimensional scaffold having a pattern formed thereon
5 is a photograph showing the degree of pattern formation according to the wavelength, frequency, pulse energy and ablation rate of the femtosecond laser
Fig. 6 is a photograph showing a pattern form according to the distance between patterns
Fig. 7 is a graph showing the physical properties of the material according to the distance between the patterns
8 is a graph showing the aspect ratio and average degree of alignment of root canal nuclei according to the distance between patterns
Fig. 9 is a photograph showing a photograph taken according to the distance between patterns by performing staining for focal adhesion on the nanofibers
Fig. 10 is a graph showing the results of the absorbance experiment for measuring the degree of cell proliferation according to the distance between patterns
11 is a photograph showing the degree of differentiation proteins of canaliculus cells on the nanofibers according to the distance between the patterns by date
12 is a graph showing the degree of alignment of canaliculus cells on the nanofibers according to the distance between the patterns
13 is a graph showing the degree of expression of Myosin Heavy Chain, which is a gene used for measurement of myoblast differentiation,
Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.
Unless defined otherwise, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, commonly used predefined terms are not ideally or excessively interpreted unless explicitly defined otherwise.
The present invention relates to a three-dimensional scaffold in which a pattern is formed and a manufacturing method thereof.
First, the three-dimensional scaffold in which the pattern of the present invention is formed may include a scaffold and a pattern formed on the scaffold.
The scaffold may comprise electrospun nanofibers. Nanofibers formed by electrospinning provide a high surface area sufficient for cell adhesion and provide excellent cell-inducing function.
The electrospun nanofiber may be selected from the group consisting of aliphatic polyesters, polyalkylene oxides, polydimethylsiloxanes, polycaprolactones, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycan, (L- or L-), lactide, poly (lactic acid), poly (lactide) glycolic acid, poly (glycolic acid), and combinations thereof. , Poly (glycolide), glycolide, poly (lactide-co-glycolide), poly (lactic acid-co-glycolic acid), and combinations thereof. More preferably, it may be poly (l-lactic acid), which has been derived from several experiments as an optimized material for the differentiation of myoblasts and the formation of muscle tissue therefrom.
In addition, it is preferable that a plurality of the patterns are formed on the scaffold surface, and the shape thereof is a groove shape.
The patterns may be arranged in various ways, but they are preferably arranged in one direction, and more preferably, arranged in parallel with each other. This is to realize a hierarchical structure of muscle tissue.
The distance between the patterns can be an average of 5 to 100 mu m, and preferably an average of 10 to 25 mu m. When the diameter is less than 5 占 퐉 or more than 100 占 퐉, the differentiation rate of the root cells is rapidly lowered, and the formed canalicules are not uniformly arranged, resulting in a problem that the average arrangement angle is significantly increased.
It is found that the distance between the patterns is important factor for the realization of the muscle tissue with hierarchical structure while facilitating the differentiation of the root cells on the scaffold composed of nanofibers, This is an optimization.
Further, the width of the pattern may be an average of 3 to 10 mu m, preferably 4 to 7 mu m, more preferably 5 to 6 mu m. When the thickness is less than 3 탆, the effect due to the pattern formation is insignificant. When the thickness exceeds 10 탆, it is difficult to implement the hierarchical muscle tissue.
The pattern can be formed in various ways, but is preferably formed by a femtosecond laser. The femtosecond laser has few defects such as surface damage and cracks, and the pattern surface is smooth and suitable for cell differentiation.
The femtosecond laser has a wavelength within 400 to 800 nm and forms the pattern at an ablation rate of 10 to 60 mm / s at a frequency of 5 to 15 kHz with a pulse energy of 0.5 to 6 μJ using a 5 to 10 magnification lens More preferably a pulse energy of 0.7 to 2 μJ at a frequency of 8 to 12 kHz and an ablation rate of 20 to 40 mm / s, most preferably a 1 μJ pulse energy at a frequency of 10 kHz and a pulse energy of 30 mm / s It is effective to form the pattern at an ablation speed of < RTI ID = 0.0 > This is an optimal condition for forming a pattern with a certain width and depth as a result of several experiments.
Next, a method of manufacturing a three-dimensional scaffold in which the pattern of the present invention is formed may include a scaffold formation step (S10) and a pattern formation step (S20) as shown in Figs.
The scaffold formation step S10 is a step of forming a three-dimensional scaffold made of nanofibers by an electrospinning method.
As shown in FIG. 3, the scaffold forming step S10 forms a three-dimensional scaffold in the form of a chamber by collecting the polymer solution into a chamber by radiating the polymer solution through a high-voltage nozzle. The shape of the chamber may be variously formed in a desired shape, but it is preferable that the shape of the shape of the chamber is a hexahedron.
The polymer solution may be at least one selected from the group consisting of polyalkylene oxide, polydimethylsiloxane, polycaprolactone, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides, aliphatic polyesters, Wherein the aliphatic polyester is selected from the group consisting of lactic acid (D- or L-), lactide, poly (lactic acid), poly (lactide) glycolic acid, poly (glycolic acid), poly (glycolide), glycolide, poly Lactide-co-glycolide), poly (lactic-co-glycolic acid), and combinations thereof.
The pattern formation step S20 is a step of forming a pattern by irradiating the femtosecond laser onto the three-dimensional scaffold. This is a process for forming an optimal pattern for muscle tissue formation and regeneration on a scaffold composed of electrospun nanofibers. As shown in FIG. 4, when a pattern is formed with a femtosecond laser as compared with other lasers, it is confirmed that there are few defects such as surface damage and cracks, and that the pattern surface is smooth and suitable for cell differentiation.
Specifically, in the pattern formation step (S20), the femtosecond laser having a wavelength within the range of 400 to 800 nm is moved in parallel in one direction, and a frequency of 5 to 15 kHz Dimensional scaffold at an ablation rate of 10 to 60 mm / s to form the pattern.
It is preferable that a plurality of the patterns are formed in a groove shape, a parallel pattern may be formed, an average distance between the patterns is 10 to 25 mu m, and an average width of the pattern is 3 to 10 mu m. The description thereof is as described above.
The pattern plays a role in controlling the adhesion and differentiation of myoblasts and thus is a key constituent for realizing a hierarchical structure of muscle tissue combined with nanofibers and nano / microsize .
On the three-dimensional scaffold after the pattern formation step, the myoblasts are differentiated to form myotubes, and the myotubes are formed hierarchically along the nanofibers and the pattern, Organization.
Here, the mean arrangement angle of the myotubes may be 1 to 15 degrees, and preferably 5 to 12 degrees. If it is less than 1 degree, economical efficiency is low. If it exceeds 15 degrees, it is difficult to be formed and regenerated as muscle tissue.
The following is an experimental result to demonstrate the superiority of the three-dimensional scaffold in which the pattern of the present invention is formed.
First, experiments were conducted on the pattern formation according to pulse energy, frequency, and ablation rate of a femtosecond laser having a pulse width of 400 fs. As shown in FIG. 5, It was confirmed that the laser of the wavelength was formed uniformly at a frequency of 10 kHz at an ablation rate of 10 to 50 mm / s, more preferably at an application rate of 30 m / s.
Further, as a result of measuring the physical properties of the pattern formed on the nanofibers at different distances between the patterns, all of the four physical properties as shown in FIG. 7 were confirmed to be maintained without adequate deterioration compared to the case without the pattern.
As a result of experiments on aspect ratio and average degree of arrangement of canalicular nuclei according to the distance between patterns, it was confirmed that they were effectively differentiated and uniformly arranged at 10 to 25 μm as shown in FIG.
Next, as shown in FIG. 10, the degree of cell proliferation and differentiation according to the distance between the patterns was measured. According to the absorbance measurement under the growth medium and the differentiation medium, when the distance between the patterns was 10 to 25 μm, And it was confirmed that the differentiation occurred actively.
As shown in FIG. 12, when the distance between the patterns is 10 to 25 μm, the canalicules are uniformly arranged in one direction, and the muscle tissue Which is similar to that of
As shown in FIG. 14, when the distance between patterns is 10 to 25 탆, the gene suitable for differentiation of myoblasts is selected as a result of measuring the degree of expression of Myosin heavy chain in differentiation of myoblasts, It can be confirmed that it is fully expressed.
Thus, through the above experiments, it was possible to differentiate stem cells and form canalic cells suitable for the formation and regeneration of muscle tissue in a three-dimensional scaffold having a specific pattern in the present invention, and its critical significance has been proved.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is clear that the present invention can be suitably modified and applied in the same manner. Therefore, the above description does not limit the scope of the present invention, which is defined by the limitations of the following claims.
Claims (20)
The scaffold is composed of electrospun nanofibers,
The pattern is in the form of a groove,
The distance between the patterns is in the range of 5 to 100 mu m on average,
The width of the pattern is 3 to 10 mu m on average,
The pattern controls the adhesion and differentiation of myoblasts. A patterned three-dimensional scaffold.
Wherein the distance between the patterns is in the range of 10 to 25 mu m on average.
Wherein the pattern comprises a pattern arranged in parallel with each other.
Wherein the pattern is a pattern formed by a femtosecond laser.
The femtosecond laser has a pulse width of less than 1000 fs, a wavelength in the range of 400 to 800 nm, and a pulse energy of 0.5 to 6 μJ using a 5 to 10 magnification lens at an ablation rate of 10 to 60 mm / s at a frequency of 5 to 15 kHz And a pattern for forming the pattern is formed.
The electrospun nanofiber may be selected from the group consisting of aliphatic polyesters, polyalkylene oxides, polydimethylsiloxanes, polycaprolactones, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycan, And a polymer selected from a combination thereof.
The aliphatic polyester may be selected from the group consisting of lactic acid (D- or L-), lactide, poly (lactic acid), poly (lactide) glycolic acid, poly (glycolic acid), poly (glycolide), glycolide, Co-glycolide), poly (lactic acid-co-glycolic acid), and combinations thereof.
And a pattern forming step of irradiating the femtosecond laser onto the three-dimensional scaffold to form a pattern having an average distance of 5 to 100 mu m between the patterns and an average width of 3 to 10 mu m of the pattern,
The pattern formation step may be performed by moving a femtosecond laser having a wavelength within a range of 400 to 800 nm in parallel in one direction and irradiating a laser beam having a pulse energy of 0.5 to 6 J using a 5 to 10 magnification lens at a frequency of 5 to 15 kHz at a frequency of 10 to 60 mm / And irradiating the pattern onto the three-dimensional scaffold at an ablation speed to form the pattern.
The scaffold forming step may include forming a pattern for forming a three-dimensional scaffold in the chamber shape by collecting the polymer solution into a chamber by radiating the polymer solution through a nozzle to which a high voltage is applied.
The polymer solution may be at least one selected from the group consisting of polyalkylene oxide, polydimethylsiloxane, polycaprolactone, polylysine, collagen, laminin, fibronectin, elastin, alginate, fibrin, hyaluronic acid, proteoglycans, polypeptides, aliphatic polyesters, Wherein the aliphatic polyester is selected from the group consisting of lactic acid (D- or L-), lactide, poly (lactic acid), poly (lactide) glycolic acid, poly (glycolic acid), poly (glycolide), glycolide, poly (Lactide-co-glycolide), poly (lactic acid-co-glycolic acid), and combinations thereof.
In the pattern formation step, a plurality of patterns are formed in a groove shape, and a parallel pattern is formed.
Wherein the distance between the patterns is in the range of 10 to 25 mu m on average.
Wherein the pattern is a pattern for controlling adhesion and differentiation of myoblasts.
Wherein a pattern in which a myoblast is differentiated to form a myotube is formed on a three-dimensional scaffold through the pattern formation step.
Wherein the average arrangement angle of the myotubes is 1 to 15 degrees.
Wherein the myotube is hierarchically formed along the nanofibers and the pattern to realize a muscle tissue.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109211974A (en) * | 2018-08-07 | 2019-01-15 | 哈尔滨商业大学 | Thermal insulation layer construction debonding defect pulsed femtosecond laser pumping infrared thermal wave detection device and method |
| KR20200067263A (en) * | 2018-04-09 | 2020-06-12 | 광주과학기술원 | Micropatterned conductive hydrogel and method for producing the same |
| KR20200075936A (en) * | 2018-12-12 | 2020-06-29 | 가천대학교 산학협력단 | Method for preparing or tube type scaffold |
| CN116065301A (en) * | 2022-12-26 | 2023-05-05 | 深圳大学 | Perovskite polymer fiber film and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200067263A (en) * | 2018-04-09 | 2020-06-12 | 광주과학기술원 | Micropatterned conductive hydrogel and method for producing the same |
| KR102250792B1 (en) * | 2018-04-09 | 2021-05-11 | 광주과학기술원 | Micropatterned conductive hydrogel and method for producing the same |
| CN109211974A (en) * | 2018-08-07 | 2019-01-15 | 哈尔滨商业大学 | Thermal insulation layer construction debonding defect pulsed femtosecond laser pumping infrared thermal wave detection device and method |
| KR20200075936A (en) * | 2018-12-12 | 2020-06-29 | 가천대학교 산학협력단 | Method for preparing or tube type scaffold |
| KR102253755B1 (en) | 2018-12-12 | 2021-05-20 | 가천대학교 산학협력단 | Method for preparing or tube type scaffold |
| CN116065301A (en) * | 2022-12-26 | 2023-05-05 | 深圳大学 | Perovskite polymer fiber film and preparation method thereof |
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