TWI421339B - Method of fabricating three dimensional scaffolds and device thereof - Google Patents

Description

Method and device for preparing three-dimensional stent

The invention relates to a method for preparing a three-dimensional stent and a device thereof, in particular to a method for preparing a three-dimensional stent for tissue engineering or cell culture and a device thereof.

Today, although biomedical research and industry are well developed, many of the ills that were previously considered to be incurable are also cured in the next breakthrough in knowledge and technology. However, for those derived from innate or acquired factors In patients with organ tissue defects or loss of function, current medical laws still rely on passive medical methods such as autologous transplantation or organ donation, and cannot propose any active and reliable treatment planning.

In order to solve this problem, experts and scholars have devoted themselves to various aspects of research, among which regenerative medicine is the largest. The purpose of regenerative medicine is to use healthy cells to replace damaged or dysfunctional cells for the purpose of saving patients. Cell culture and tissue engineering are the key technologies in regenerative medicine. They are multiplied by human means to provide sufficient cells for experimental analysis, and then simulate and provide the conditions required for tissue cell growth and development. It can grow into cells or tissues with specific properties, and even form organs with complete functions.

Although the use of regenerative medicine can not only solve the uncertainty of waiting for organ donation, but also eliminate the potential doubts that the immune system may be rejected after organ transplantation. However, its development has been limited by a few technologies, such as cell culture and stereo scaffolds.

Conventional cell culture techniques are mostly cultured on plastic petri dishes. Therefore, when the number of cells is increased to a certain amount, the cells will overlap each other and lack growth space. Therefore, cell culture can only be performed on a two-dimensional plane. The progress is not only the frequency of treatment, but also the amount of cells that can be produced per culture is very limited, which greatly affects the development of further research experiments such as tissue engineering.

In addition to cell culture, the use of scaffolds plays an extremely important role in order to allow cells grown through culture to eventually grow into tissues or organs that conform to ideal functions and patterns. The function of the scaffold is to provide a three-dimensional framework suitable for cell growth, which is generally referred to as a three-dimensional scaffold, which has a large number of holes for cell attachment or inoculation, thereby guiding the cells to grow and differentiate in a three-dimensional direction according to the plan. Produce a pseudo-regenerative tissue or organ. Conventional stent preparation techniques include a salting-out process, a freeze drying process, and a solid freeform fabrication process.

However, in practice, such preparation techniques generally have considerable bottlenecks, such as the necessity of using expensive machine equipment for control or the preparation process, which takes time and time. Moreover, organic solutions that remain on the three-dimensional scaffold during certain preparation steps can also cause damage to the cells. In addition, the stents prepared by the conventional methods have great differences in the size and shape of the internal pore size, which brings many experimental and research uncertainties, which indirectly hinder cell culture and tissue engineering. development of.

In view of this, how to provide a method for preparing a three-dimensional stent to prepare a stent suitable for cell culture and tissue engineering, and at the same time saving the cost and process time of manufacturing the stent is one of the current important issues.

In view of the above problems, an object of the present invention is to provide a method and a device for preparing a three-dimensional stent, which can form a stereo stent suitable for cell culture and tissue engineering, and simultaneously increase cell acquisition and save. The cost of making the rack and the time it takes to make the process.

To achieve the above object, a method for preparing a three-dimensional stent according to the present invention comprises: forming a plurality of bubbles by passing a substrate solution and a gas stream through a bubble generator; and performing a gel reaction on the bubbles.

To achieve the above object, a three-dimensional scaffold preparation apparatus according to the present invention comprises a bubble generator, a substrate solution supply unit, and an air flow supply unit. The bubble generator comprises an outer tube and a inner tube, wherein the outer tube has a first end gradually decreasing in diameter, the inner tube is sleeved in the outer tube, and the second end is gradually reduced in diameter. The matrix solution supply unit is connected to the outer tube. The air supply unit is connected to the inner tube.

Further, in the method of producing a three-dimensional scaffold of the present invention, the bubble sizes formed are substantially equal to each other.

As described above, according to the method and apparatus for preparing a three-dimensional stent according to the present invention, it is only necessary to use a bubble generator to form a plurality of bubbles in the flowing matrix solution and the gas stream, and then to form a bubble for gel formation. The engineered cell scaffold is a simple and fast way to prepare a three-dimensional scaffold. In addition, the apparatus for preparing a three-dimensional stent of the present invention has a simple structure and is easy to operate, can be used without excessive mental effort, and is easy to carry and assemble, and is advantageous for the promotion of tissue engineering technology. Furthermore, the matrix solution used in the method for preparing the three-dimensional scaffold of the present invention can be easily replaced according to the needs of the user, thereby producing bubbles of different sizes, thereby customizing a specific three-dimensional scaffold. In some embodiments of the present invention, the stent prepared according to the method for preparing a three-dimensional stent according to the present invention can have pores of substantially the same size and shape under the control of appropriate conditions, thereby avoiding structural differences between the pores in the stent. Influence the results of the analytical study. Compared with the prior art, the method and device for preparing the three-dimensional stent of the present invention can replace the complicated working process and expensive mechanical equipment, and have no doubt that the harmful organic solvent is used, so the method for preparing the three-dimensional stent of the present invention is utilized. The resulting cell culture and tissue engineering scaffolds not only simplify the process, but also reduce application costs.

Tissue engineering is the core technology for regenerative medicine. It is mainly composed of multiple programs. The outline is to process the appropriate biomedical materials to form a three-dimensional basic structure, which is the term "three-dimensional scaffold". Then, the selected cells are attached, or perfused or inoculated onto the scaffold, or the three-dimensional scaffold itself is the medium of the cells, allowing the cells to grow in the scaffold. After that, the cells are given appropriate growth signals and chemical stimulation, so that the cells can proliferate, grow and differentiate under the simulated environment, and then form a regenerative tissue or organ that is intended to be treated as a therapeutic target. When transplanted into the patient, it can replace the original one. Damage, or dysfunctional or necrotic tissue and organs for medical purposes.

The preparation method of the three-dimensional scaffold of the present invention can be applied to the above-defined tissue engineering field after careful consideration, and accordingly, any steps or procedures identical or similar to those described above are used to achieve the same or similar medical purposes. They are all included in the term "tissue engineering" as used herein, so that the three-dimensional scaffold or scaffold used or the functionally equivalent to the three-dimensional scaffold is similar or identical to the preparation of the three-dimensional scaffold of the present invention. The object to which the method applies.

However, it should be noted that the application of the tissue engineering three-dimensional scaffold is only a non-limiting embodiment of the preparation method of the present invention, and other modes of application, such as providing a three-dimensional structure for cell culture, are also the present invention. The object to which the preparation method is applied.

The term "scaffold" as used in the present invention is a three-dimensional structure comprising any shape, size or composition, and can be used as at least one structure for attaching, attaching or implanting cells, and achieving normal growth and/or proliferation of cells. And/or the purpose of differentiation. In an embodiment of the present invention, since the three-dimensional scaffold made by using the preparation method and the apparatus disclosed by the present invention is guided by medical use, it is still preferably bio-inclusive in use, and implanted in the living organism. After the body will gradually degrade, to avoid the burden of the organism. In another embodiment of the present invention, the three-dimensional scaffold made by the preparation method and apparatus disclosed in the present invention is used for cell culture, and has high gas permeability and nutrient permeability (ie, has a preferred specific surface area). ).

In one embodiment of the present invention, a three-dimensional scaffold made by the preparation method and apparatus of the present invention is used to attach or implant cells for normal growth, and/or proliferation and/or differentiation. The cell type may be any naturally obtained cell, or a human bioengineered cell or both of them, and there is no particular limitation on the use, but if the subsequent application value is considered, the cell is preferably A monopotent stem cell, or a pluripotent stem cell, or a totipotent stem cell or a cancer cell strain. In detail, for example, but not limited to, embryonic stem cells, cord blood stem cells, hematopoietic stem cells, mesenchymal stem cells, and endothelium Endogenous progenitor cells, dental pulp stem cells, human cervical epithelial cell lines (HeLa cells), and human colorectal cancer cell lines (HCT116 cells); however, other cells that are easy to culture may also Use, for example, fibroblasts. In one embodiment of the invention, the method of preparation of the present invention and apparatus therefor are for producing a three-dimensional scaffold suitable for cell culture, and the cells attached thereto, or implanted or perfused thereon, are fibroblasts.

Hereinafter, a method of preparing a three-dimensional stent of the present invention will be described with reference to the related drawings, in which the same elements will be described with the same reference numerals.

Please refer to FIG. 1 , which is a flow chart of steps of a method for preparing a three-dimensional stent disclosed in the present invention. The method for preparing a three-dimensional stent disclosed in the present invention comprises the steps of: forming a plurality of bubbles (S20) by passing a substrate solution and a gas stream through a bubble generator; and performing a gel reaction (S40) on the bubbles.

In the step S20, the substrate solution M is not particularly limited in selection, and may be a solution, or a suspension or a mixed solution, and it is preferable to form a suitable bubble with the air flow A to form a suitable bubble. In the present embodiment, the matrix solution M is an aqueous solution added to the substrate, wherein the substrate may be a biodegradable biopolymer, or a degradable synthetic polymer or a non-degradable synthetic polymer. In the case of biodegradable biopolymers, for example, but not limited to, gelatin, argarose, hyaluronic acid, chitosan, and alginate, And collagen, fibrin, and cellulose. The degradable synthetic polymer can be, for example but not limited to, polyester amide (for example, BAK 1095 or polyester decyl C), and polycaprolactone (PCL), and polylactic acid (poly). Lactic acid, PLA) (for example Type polylactic acid) and methoxy-terminated poly(ethylene oxide). As for the non-degradable synthetic polymer, for example, but not limited to, poly(dimethylsiloxane), PDMS, and thermoplastic polyurethane (for example) 950), and polyethylene (poly (ethylene terephthalate), PET), and polytetrafluoroethylene (PTFE) and poly(vinylidene fluoride), PVDF ). Of course, the above is only an example, and any one that achieves the same purpose and effect as described above can be used as a substrate.

After the addition of the above substrate, the matrix solution M can be made slightly thick to contribute to bubble formation. As for the content of the matrix in the matrix solution M, it can be adjusted depending on the formation of the bubble. In general, the matrix content in the matrix solution M is about 10% by weight. In one embodiment of the invention, the matrix solution M is comprised of 1% to 2% seaweed gum. In yet another embodiment of the invention, the matrix solution M is comprised of 6% to 10% gelatin. In addition, in order to enhance the preparation of the stent, in the present embodiment, the matrix solution M may further contain a surfactant, and the content of the surfactant may also be adjusted according to the requirements for forming bubbles, in an embodiment of the present invention. The matrix solution M may contain a biocompatible surfactant, for example, 1% by weight. F127. Additionally, in one embodiment of the invention, the gas stream A is preferably formed by a flow of a chemically stable gas such as, but not limited to, a nitrogen gas stream or an air stream.

In one embodiment of the present invention, the three-dimensional scaffold produced by the method for preparing a three-dimensional scaffold of the present invention is prepared for cell culture and/or tissue engineering, so that any possible risk of contamination must be reduced. Accordingly, in the present embodiment, the method for preparing the three-dimensional scaffold of the present invention may further include a step S10 of filtering or sterilizing the matrix solution M and the gas stream A to remove the matrix solution M or the gas stream A before the step S20. Microorganisms, or spores or harmful substances present, to avoid the formation of three-dimensional scaffolds with contamination or potential contamination risks, affecting subsequent cell culture and/or tissue engineering.

Referring to FIG. 1 and FIG. 2 simultaneously, the present invention further provides a three-dimensional stent preparation device 4 comprising a bubble generator 1, a substrate solution supply unit 2 and an airflow supply unit 3. In step S20 of the preparation method, the matrix solution M and the gas stream A are passed through the bubble generator 1 to form a plurality of bubbles, and the bubble generator 1 can form the bubble of the flowing matrix solution M and the gas stream A, so that any one can be achieved. The device or apparatus of the present invention can be used as the bubble generator 1 of the present invention, and there are many variations in the structure. Referring to FIG. 2, in an embodiment of the present invention, the bubble generator 1 can be a bubble generator 1 having a double tube structure, and in detail, is a double capillary or double needle tube structure, which comprises a The outer tube 11 and an inner tube 12 are sleeved in the outer tube 11, and the outer tube 11 is for the matrix solution M to flow, and the inner tube 12 is for the gas A to flow. The manner in which the inner tube 12 is sleeved in the outer tube 11 is not limited, but it is preferred that both the outer tube 11 and the inner tube 12 have the same center in the same plane cross section after the sleeve is completed.

In this embodiment, the outer tube 11 and the inner tube 12 are made of metal, or glass or plastic. However, to create a suitable cell growth environment, the bubble generator 1 has a bubble diameter of between about 50 microns and 500 microns, and a preferred cell diameter of between about 50 microns and 100 microns. Further, the tips of the outer tube 11 and the inner tube 12 are preferably formed by heating and melting a columnar glass capillary tube to form an outer tube 11 and an inner tube 12 made of glass. In this embodiment, the outer tube 11 is drawn by a square capillary tube, and the inner tube 12 is drawn by a cylindrical capillary tube. After the completion of the system, both ends of the bubble are formed into a cylindrical shape. Furthermore, referring to FIG. 2, in the present embodiment, the outer tube 11 has a first end 111 whose diameter is gradually reduced, and the inner tube 12 has a second end 121 whose diameter is gradually reduced. Therefore, the outer tube 11 and the inner tube 12 are similar in appearance to the shape of a dropper, and have a pointed tip which is relatively thin with respect to other portions of the column. The use of a capillary tube having a small size for the capillary facilitates the generation of smaller sized bubbles. In addition, in order to form a suitable bubble, the cross-sectional shape of the outer tube 11 and the inner tube 12 is preferably the same, and the diameter of the outer tube 11 is only slightly larger than the diameter of the inner tube 12.

The substrate solution supply unit 2 is used for providing the matrix solution M required for the bubble generator 1 to form a bubble, and may be any one that can easily replenish the matrix solution M and circulate the matrix solution M to the outer tube 11, which may be, for example, but not It is limited to a tank having a liquid storage function, and is not particularly limited in structure or shape. The substrate solution supply unit 2 preferably maintains the minimum contamination probability of the internal matrix solution M and has an output pressure and flow rate of the adjustable matrix solution M. / or unit time output and other functions.

The airflow supply unit 3 is for providing the airflow A required for the bubble generator 1 to form a bubble. Similarly, the airflow supply unit 3 can be any one that can easily fill the gas and circulate the gas to the inner tube 12 in the form of the airflow A, which can be, for example but not limited to, a tank having a gas storage function, and has no special structure or shape. limit. Among them, the airflow supply unit 3 preferably has a function of maintaining a minimum pollution probability of the internal gas and having a discharge pressure, a flow velocity, and/or a gas output per unit time of the adjustable airflow A.

In order to ensure that the formed stent is free from contamination, please refer to FIG. 2, in the present embodiment, the bubble generator 1 is transported to the outer tube 11 and the inner tube 12 by the substrate solution supply unit 2 and the air supply unit 3, and even In the last circulation path to bubble formation, it is a closed pipeline or passage, which can achieve the purpose of reducing the risk of pollution.

Referring to FIG. 1 again, in the embodiment, in the process of preparing the three-dimensional support, it is also necessary to consider the formation environment parameters and operating conditions of the air bubbles, wherein the environmental parameters and the operating conditions cover the range covered by the matrix solution supply unit 2 The air supply unit 3 is included in the outer tube 11 and the inner tube 12, for example, the flow rate of the matrix solution M, and the discharge pressure of the air stream A, and the tube diameter and nozzle size of the outer tube 11 and the inner tube 12 in the bubble generator 1. And the relative position of the nozzle between the outer tube 11 and the inner tube 12. In other words, in the present embodiment, the relevant environmental parameters and operating conditions that affect the bubble generation are controlled to form a suitable bubble, wherein the flow pressure A from the inner tube 12 and the flow rate of the substrate solution M It is a key influencing factor and should be properly designed and controlled.

It is particularly emphasized that adjusting the environmental parameters and operating conditions of the bubble generator 1 in combination with the substrate solution M and the gas stream A used can achieve a preferred parameter setting for forming bubbles. Under this setting, the sizes of the formed bubbles can be made substantially equal, and these bubbles are also suitable bubbles for use in the three-dimensional scaffold preparation method of the present invention. In the present embodiment, the preferred environmental parameter is that the inner diameter of the first end 111 of the outer tube 11 is about 62 to 75 micrometers (μm), and the inner diameter of the second end 121 of the inner tube 12 is about 25 to 35 microns, the second end 121 of the inner tube 12 has an inner diameter of about 350 to 450 microns with respect to the other side of the nozzle, and the outer tube 11 is spaced from the inner tube 12 with a nozzle of about 350 to 450 microns ( The inner tube 12 is more retracted).

As shown in FIG. 3, it is a relationship diagram between the operation setting of the bubble generator 1 and bubble formation. The control of the operating conditions of the bubble generator 1 is mainly achieved by changing the flow rate of the substrate solution M and the discharge pressure of the gas stream A. In Fig. 3, each mark point is an experimental result of different substrate solution flow rates and different gas flow discharge pressures, and the circular solid/hollow mark points indicate that bubbles of uniform size are generated; the triangle solid/hollow mark points indicate that two different sizes of bubbles are simultaneously generated. Cross-marking dots indicate that bubbles cannot be generated; star-shaped dots indicate that bubbles of different sizes are simultaneously produced. In addition, the straight line L1 indicates the boundary between the bubble formation and the liquid. In the region above the straight line L1, the matrix solution M flows out in the form of a liquid, and no bubble can be formed; the broken line L2 represents the boundary where the formed bubble can be stably existed. However, in the region below the dotted line L2, bubbles are not stably generated or the generated bubbles are not stable and fragile, that is, the operation setting between the straight line L1 and the broken line L2 is an operation suitable for bubble formation. set up. It ranges from about 300 microliters per minute (μL/min) to 1,050 microliters/minute of the substrate solution M; and the gas stream A discharge pressure is in the range of from about 3 Pascals (psi) to 10 Pascals. Preferably, when the substrate solution M has a flow rate of 500 microliters and a pressure of 5.96 pascals, the bubble generator 1 can produce bubbles of substantially uniform size. In this way, the results of the analytical study can be avoided due to structural differences between the holes in the stent.

Referring to FIG. 1 again, the method for preparing the three-dimensional stent of the present invention may further comprise a step of collecting bubbles (S30). In step S30, a container may be placed under the bubble generator to receive the newly formed bubbles. And during the process of collecting bubbles, the bubbles are automatically stacked in a tight arrangement (as shown in Figures 4A, 4B and 4C).

Referring to FIG. 1 again, after the step S30 of collecting bubbles, the method for preparing the three-dimensional stent of the present invention may further comprise performing a gel reaction on the bubbles (S40). In step S40, the gel reaction is a chemical or physical change between the bubble and the bubble, so that the bubble wall and the bubble wall of the bubble are connected to each other, and gelation occurs to fix the adjacent two bubbles. Relative position, forming a three-dimensional stent. From a giant point of view, the gel reaction can form a plurality of bubbles that are originally stacked, preferably a bubble wall that is automatically stacked into closely packed plural bubbles, and a three-dimensional colloidal structure is formed from a thick liquid matrix solution, that is, three-dimensional. support. In one embodiment of the invention, the wall of the bubble that completes the gel reaction is a sponge-like or honeycomb-like structure with a large number of pores suitable for attachment or seeding of cells. The conditions for performing the gel reaction differ depending on the substances contained in the matrix solution M. For example, if the matrix solution M contains alginate, gelation can occur by cross-linking, for example by adding an appropriate amount of a divalent metal ion (e.g., calcium or magnesium). In one embodiment of the invention, for example, a calcium chloride solution having a concentration of 100 millimolar or higher is added. On the other hand, if the matrix solution M contains gelatin, the gel reaction is carried out under the condition that the temperature is lowered before the chemical cross-link is performed. If the matrix solution M contains chitin or collagen, the gel reaction conditions are to adjust the pH to neutral and simultaneously increase the temperature, and it is preferred to raise the temperature by more than 37 °C. Of course, in the case of blending the substance contained in the matrix solution M, other ones which can perform a gel reaction or achieve the same purpose as the gel reaction can also be used in this step.

In addition, in an embodiment of the present invention, the method for preparing a three-dimensional stent of the present invention may further comprise the step of removing the gas in the bubble (S50) after the step S40 of performing the gel reaction on the bubble. Wherein, the gas in the bubble is preferably removed by suction in a low pressure environment or in a vacuum environment; in detail, the bubble is immersed in a liquid in a low pressure environment and then pumped in a pumping manner. In addition to the gas in the bubble, the gelled structure formed by the gelation of the bubble can be immersed in the liquid. Since the gas pressure of the external environment is less than the pressure of the gas in the bubble in the solidified structure, the gas will automatically be removed from the stent. The medium is released, and the gap between the entire stent is filled by the liquid to achieve the purpose of preserving the stent. Among them, the liquid to be used may be, for example, general pure water or deionized water. Of course, other liquids which are chemically stable and compatible with the cured structure may be selected without particular limitation. It should be noted that in the step S50 of removing the gas in the bubble, it is preferable that the void inside the stent to be gelatinized is completely filled with the liquid to ensure that the gas in the bubble is surely removed.

Please refer to FIG. 5 and FIG. 6. FIG. 5 is a structure observed under a scanning electron microscope after the three-dimensional stent is sliced, and FIG. 6 is a three-dimensional stent observed under a scanning electron microscope. structure. As can be seen from the figure, the stent is a three-dimensional structure having a large number of holes, and the shape of the stent is substantially regular, and the size of the holes formed by the stent is substantially the same size.

In one embodiment of the present invention, the three-dimensional scaffold prepared by the method for preparing the three-dimensional scaffold of the present invention can be stored in a 0.5 M calcium ion or magnesium ion solution supplemented with 1% antibiotic, and stored at 4 ° C to Avoid microbial contamination and remove it when cells are inoculated.

In one embodiment of the present invention for human cells are seeded fibroblasts in culture until the number of fibroblasts was greater than ^106, the stent can first culture solution in the 12-well plates Run Wash twice, then attach or inoculate the cells on the three-dimensional scaffold by injection to further culture the cells on the three-dimensional scaffold (as shown in Figure 7, where there are round dots and slightly fusiform) For fibroblasts, the rest of the irregular triangles are three-dimensional scaffold structures). It should be noted that in the preparation method of the three-dimensional scaffold of the present invention, the attachment or seeding of the cells is not necessarily performed after the preparation of the stent is completed, and the matrix solution M and the gas stream A may be flowed through the bubble generator 1 to form. Prior to the step S20 of the plural bubbles, the cells are previously injected, or cultured or mixed in the matrix solution M to complete attachment or inoculation. The scaffold prepared in this manner can directly follow the procedure of subsequent cell growth and differentiation after the scaffold is formed. That is to say, in the present invention, when the cells to be cultured are transplanted or inoculated into the three-dimensional scaffold, the cells may be mixed with the matrix solution constituting the three-dimensional scaffold material at the beginning to allow the cells to start growing inside the three-dimensional scaffold. Another timing is when the three-dimensional scaffold is prepared, and then the cells, or the cell suspension or the cells mixed with the medium are inoculated onto the three-dimensional scaffold, and the cells are obtained on the surface of the three-dimensional scaffold or the holes formed by the three-dimensional scaffold. Growing in.

In summary, according to the method for preparing a three-dimensional stent according to the present invention, it is only necessary to use a bubble generator to form a plurality of bubbles in the flowing matrix solution and the gas stream, and then to form a bubble gel to form a cell culture and/or Or a tissue-engineered three-dimensional stent is a simple and quick way to prepare a stent. In addition, the matrix solution used in the method for preparing the three-dimensional scaffold of the present invention can be easily replaced according to the needs of the user, so that bubbles of different sizes can be produced, thereby customizing a specific stereo scaffold. In some embodiments of the present invention, the three-dimensional scaffold made by the method for preparing the three-dimensional scaffold according to the present invention can have holes with substantially the same size and shape by appropriate condition control, thereby avoiding structural differences between the holes in the scaffold. Influence the results of the analytical study. Compared with the prior art, the preparation method of the three-dimensional stent of the present invention is simple in operation and has no doubt that harmful organic solvents are used, so that the three-dimensional stent prepared by the preparation method of the three-dimensional stent of the invention can not only simplify cell culture or tissue. The process of the rack that can be used in the project can reduce the cost in the production process.

The invention also discloses a device for preparing a three-dimensional stent, which is a device which is simple in structure, light and easy to assemble, and has wide popularity and convenient use compared with the expensive mechanical equipment used for fabricating tissue engineering stents. High sexuality.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

1. . . Bubble generator

11. . . Outer tube

111. . . First end

12. . . Inner tube

121. . . Second end

2. . . Matrix solution supply unit

3. . . Air supply unit

4. . . Three-dimensional stent preparation device

A. . . airflow

M. . . Matrix solution

L1. . . straight line

L2. . . dotted line

S10~S50. . . step

1 is a flow chart of a method for preparing a three-dimensional stent according to a preferred embodiment of the present invention;

Figure 2 is a schematic view of the bubble generator in step S20;

3 is a diagram showing experimental data of operation setting of bubble formation in the bubble generator in step S20;

4A and 4B are photographic views of the bubble stack in step S30;

Figure 4C is a partial enlarged view of Figure 4B;

5 is a cross-sectional view of a stent prepared by a scanning electron microscope according to a method for preparing a three-dimensional stent according to a preferred embodiment of the present invention;

6 is a perspective view of a stent prepared by a scanning electron microscope according to a method for preparing a three-dimensional stent according to a preferred embodiment of the present invention;

Figure 7 is a perspective view of a three-dimensional scaffold prepared by inoculating cells and culturing the preparation method of the three-dimensional scaffold according to the preferred embodiment of the present invention.

S10~S50. . . step

Claims (21)

A method for preparing a three-dimensional stent comprises: forming a plurality of bubbles by a matrix solution and a gas flow through a bubble generator; collecting the bubbles, and stacking the bubbles into a tight arrangement; performing a gel reaction on the bubbles; And removing the gas in the bubbles, wherein the bubble generator comprises an outer tube and an inner tube, the inner tube is sleeved in the outer tube, and the matrix solution flows through the outer tube, The air flow is passed through the inner tube. The preparation method of claim 1, wherein the substrate solution comprises gelatin, acacia, hyaluronic acid, chitin, algin, cellulose or collagen. The preparation method of claim 1, wherein the substrate solution contains a surfactant. The preparation method according to claim 1, further comprising: filtering or sterilizing the substrate solution. The preparation method of claim 1, wherein the gas stream is a nitrogen gas stream or an air stream. The preparation method of claim 1, wherein the outer tube and the inner tube are made of metal, or glass or plastic. The preparation method according to claim 1, wherein the outer tube has a square cross section, and the inner tube has a circular cross section. The preparation method according to the first aspect of the patent application, wherein the gas is The dimensions of the bubbles are substantially equal. The preparation method of claim 1, wherein the diameter of the bubbles is between 50 micrometers and 100 micrometers. The preparation method of claim 1, wherein the gel reaction is produced by adding a divalent metal ion, or adjusting the temperature or adjusting the pH. The preparation method of claim 1, wherein removing the gas system in the bubbles removes the bubbles in a low pressure environment. The preparation method according to claim 11, wherein the bubbles are immersed in a liquid in a low pressure environment to evacuate the gas in the bubbles. A device for preparing a three-dimensional stent comprises: a bubble generator, comprising: an outer tube having a first end gradually decreasing in diameter; and an inner tube disposed inside the outer tube and having a diameter gradually decreasing a second end; a substrate solution supply unit coupled to the outer tube; and a gas flow supply unit coupled to the inner tube. The preparation device according to claim 13, wherein the bubble generator is a double capillary structure or a double needle structure. The preparation device of claim 13, wherein the first end and the second end have the same center on the same cross section. The preparation device of claim 13, wherein the The inner diameter of the nozzle at one end is 62 to 75 microns. The preparation device of claim 13, wherein the second end has an inner diameter of 25 to 35 μm. The preparation device of claim 13, wherein the nozzle of the first end is spaced from the nozzle of the second end by 350 to 450 microns. The preparation device of claim 13, wherein the second end is 350 to 450 microns in inner diameter of the tube on the other side of the nozzle. The preparation device of claim 13, wherein the outer tube and the inner tube are made of metal, or glass or plastic. The preparation device according to claim 13, wherein the outer tube has a square cross section, and the inner tube has a circular cross section.