TWI233943B - Method for forming scaffold - Google Patents

Abstract

The present invention discloses a method for forming scaffold. This invention provides various fixation agents for different polymers or applications, and a fixation process is performed by the fixation agent at low temperature. Moreover, the method comprises a dissolution process, a temperature adjusting process, a freezing process and fixation process, wherein the fixation process is selected from the group of a solid-liquid exchanging process, a neutralization process or a gelation process.

Description

1233943 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for forming a biological substrate, and more particularly, to a method for forming a biological substrate under a low temperature condition. [Previous technology] Tissue engineering is one of the fields that have flourished in recent years. One of its famous application methods is to implant cells into biodegradable materials and wait for the cells to grow into functional new tissues or organs. After being implanted into the human body, tissue workers have become a popular method for treating organ function shortage or tissue necrosis. The research on tissue engineering can be roughly divided into three parts: cells, biocompatible substrates (scaffolds), and signals that stimulate cells to show specific behaviors. They influence each other and jointly regulate the differentiation and performance of tissues. The role of 'biological substrates' in tissue engineering includes: 1. Transporting cells or tissues that have been initially cultured to specific parts of the body, avoiding cultivated Cells or tissues are prematurely damaged by immune or other physical effects in the body. 2 · Make the newly generated tissues or organs have a three-dimensional shape that meets the requirements, and give them support and protection. · 13 1233943 3. Stimulate cells or tissues to show specific behaviors, such as coating the surface of a biological substrate with a substance that can produce a signal to achieve the desired effect. The methods of manufacturing biological substrates can generally be divided into phase separation methods and non-phase separation methods. The state of the molecular solution in 5 weeks, the position of the solution in the thermodynamic phase diagram changes, and it enters the phase separation ( In the region of separation), effective manufacturing methods to achieve the preparation of holes include temperature induced (thermally induced phase separation), adding a non-solvent, and changing the composition of the solution. Common methods for making non-phase separation procedures include particle filtration of pore former, 3D printing, high-pressure gas or supercritical fluid gas foaming, and sintering (s-g). Molten polymer particles. Generally speaking, the most common method in the above-mentioned phase separation method for manufacturing biological substrates is cold; Dong drying method, cold; Dong drying method can be roughly divided into two steps: cold> dong and drying ' The polymer solution reduces the temperature, so that it changes from the original homogeneous to a heterogeneous phase. This situation is called phase separation. The polymer solution is cut into a molecular poor phase and a polymer rich phase. When the solvent is removed, the high-lean phase opens pores, and the south-rich phase forms pore walls. In other words, when the molecule solution is frozen (phase separated), the porous structure of the substrate is actually formed, so the polymer-rich phase type is the factor that determines the structure of the biological substrate _ 14 1233943. As mentioned earlier, the reason for the cold beam drying and the preparation of Ben + ^ τ μ, the organization of industrial and private substrates is that n is under the condition of low temperature (low temperature / low melting point). ), Vacuum the system ^ 卞, when the mixture is frozen, it is directly changed to gas and removed, such as ★ this original membrane structure is no longer: ring. However, there are some inherent limitations of chilling drying as follows: 1. Solvent restrictions

Under the threshold of large-scale production, the vapor pressure of the solvent will be lower than at normal temperature, which makes the difficulty of removing the solvent greatly increased, and it is also prone to the problem of solvent residue. 2. Consumption of energy: Cold power systems and vacuum pumps consume a lot of power, which is often time-consuming. Due to the reduced volatility of the solvent at low temperatures, it requires a longer period of time to remove the solvent. If it is a high-boiling solvent system For several days, the solvent is still not completely removed. 4.Instruments and equipment: Special cooling is needed; Dong drying equipment is expensive. In view of this, it is still necessary to develop a new biological substrate process to meet the needs of rapid manufacturing, energy saving and low price, and use: 口 1 > »other manufacturing cost’ to improve production efficiency. [Summary of the Invention] In view of the above background of the invention, in order to overcome the traditional shortcomings of the invention, the invention provides a new method for forming a biological substrate to meet the needs of man-made systems, speed, energy saving and low price. 15 43 One purpose of the present invention is to provide different types of fixing agents for fixing procedures under low temperature conditions according to different polymer materials or applications. Since the polymer-depleted phase is removed or the polymer-rich phase is fixed at a low temperature, the porous structure of the polymer-rich phase can be maintained and a biological substrate can be formed therefrom. Another object of the present invention is to increase the number of solvents to be used. For example, a solvent with a high Buddha point can be used, or a solvent that can not be processed by conventional cold drying and drying. As the choice of solvents used increases, the choice of high-molecular materials also increases, so it can be made with different materials ^ biological substrates. '' According to the purpose described above, the method of forming, this method has simple steps and drying equipment, and therefore, the present invention can be used. The above-mentioned biological substrate has a dissolving process and a temperature adjustment procedure. The fixing procedure further includes the following sequence, an acid test neutralization procedure, and a glue. [Embodiment] The present invention discloses a biological substrate sheet, which does not need to use a special Freezing conforms to economic benefits and industrial formation methods including the following steps: one of a sequence, a freezing procedure and a fixed procedure: a solid-liquid exchange procedure.

M heart difficult method. In order to fully understand the present invention, detailed process steps or constituent structures will be described below. To put it bluntly, the practice of the present invention is not limited to the specific details familiar to those skilled in the field of bio-substrates. The other well-known components or process steps in 1212943 are not described in detail to avoid unnecessary limitations of the present invention. The preferred embodiments of the present invention will be described in detail as follows. However, in addition to these detailed descriptions, the present invention can be widely implemented in other embodiments, and the scope of the present invention is not limited, which is subject to the scope of subsequent patents. . Referring to the first figure, in a first embodiment of the present invention, a polymer feed 110 is first provided, wherein the polymer feed is one of the following groups: Polylactic acid (PLA) , · Polylactic acid-glycolic acid [p〇ly (lactide-co-glyCoiide), PLGA], PHA [P〇ly (hydroxyalkonate) s], PHB [poly (3_hydroxybutyrate)], polycaprolactone (PoiCaproiactone, PCL) and its derivatives. Polylactic acid (pla) includes L-polylactic acid (PLLA), d-polylactic acid (PDLA), and poly-L-d-lactic acid PDLLA, and the commonly used solvent of polylactic acid (PLA) is 1,4-dioxolane (1, 4-dioxane); commonly used solvents for polylactic acid-glycolic acid (PLGA) include Dim ethyl sulfoxide (DM SO) or ethyl acetate or 1,4-dioxolane ( 1,4-dioxane) or Dimethylformamide (DMF) or N-Methyl-Pyrrolidone (NMP); The common solvent of PHA is one of the following groups: chloroform ), Acetonitrile, benzene, cyclohexane, 1212943943 (cyclohexane), 1,4-dioxane (l, 4-dioxane), and dimethyl sulfoxide (DMSO); PHB of Commonly used solvents are one of the following groups: chloroform, trichloroethylene, 222-trifluoroethanol, N, N-Dimethylformamide (DMF), ethylacetoacetate and triolein; polycaproline The common solvents of polycaprolactone (PCL) are the following groups One of: 1,4-dioxane (1,4-dioxane), Lu chloroform (chloroform), dimethylformamide (DMF) and two Yue sulfoxide (Dimethylsulfoxide, DMSO). Then, in this embodiment, a polymer feed 110 is transported to a mixing device to perform a dissolving process 1 35 to dissolve the polymer feed 1 1 0 in a dissolving solution 120. The dissolving solution contains The above-mentioned solvent forms a solution 140 with a specific viscosity, and the viscosity of the solution 140 decreases with the decrease of the amount of the polymer feed 110 to increase the prepared bio-based material. Holes. When the polymer feed 1 10 is PLA and its derivatives, the weight content range is about 11% to 15 wt% of the above solution 140. Among them, the preferred weight content range is about the above solution 1 4 〇3wt% to 10wt%; when the polymer feed 110 is plga and its derivatives, its weight content range is about 1 wt% 'to 25wt% of the above solution 1 40, where, compared with A preferred weight content range is about 3% to 10% by weight of the above solution-18 1233943 140. Next, a cold beam procedure 145 at a specific temperature is used to solidify the above-mentioned solution 140 and form a solid junction between a polymer-rich phase substance and a polymer-lean phase substance. Among them, the south molecular-lean phase can form the pore portion of the biological substrate. The pore wall portion of the biological substrate is formed. Moreover, the pores of the substrate are reduced as the specific temperature is lowered. A non-solvent 130 is supplied and an adjustment temperature program 160 is performed, and the temperature of the agent 130 is reached to a specific temperature of the freezing program 45. Following a solid-liquid exchange program 155, the above-mentioned non-solvent 13 is used to replace the tritium as a sub-lean phase, and a biological substrate is formed. 6 5 In this embodiment, before the freezing procedure 1 45, a defoaming procedure is further performed. In order to remove the air bubbles contained in the solution, the freezing process 145 described above further includes a pre-drying process. This sequence is performed at the specific temperature mentioned above to carry out the polymer-lean phase substance in the 卩 κ 卩 卩 structure surface. On the other hand, in the sequence 160, the above-mentioned non-solvent 13 is still at a specific temperature, or the non-solvent 130 further includes an antifreeze so as not to be able to maintain a liquid state at a specific temperature. When the above polymer progresses to PLA and its derivatives, the non-solvent i 30 is a one-liquid 'wherein' the ethanol concentration in the ethanol aqueous solution ranges from wt% to just Wt%, and its preferred ethanol concentration is The scope is one structure for the next! 50, while the high score is made of the second, the adjustment of non-dissolved, carried out in exchange and 〇 contains the implementation. In addition, the pre-drying temperature range of 150 can maintain the liquid solvent of 1300, 1100, and 100% ethanol. The water-soluble ethanol is 605.5% by weight, 1233943 to 85% by weight. When it is plga and its derivatives, its non-solvent 130 is an ethanol aqueous solution, wherein the ethanol concentration contained in the ethanol aqueous solution ranges from 20 wt% / 50 to 50 wt%. The ethanol concentration range was 25 wt% to 35 wt%. . Referring to the second figure, in a second embodiment of the present invention, a polymer feed 210 is first provided, wherein the polymer feed 21 described above further includes a chitosan and a derivative thereof. Then, the polymer feed 2 10 is transported to a mixing device to perform a dissolution process 2 3 5 to dissolve the polymer feed 2 10 in a dissolving solution 220. The dissolving solution contains the above-mentioned solvent and forms The first solution 240 having a specific viscosity, and the viscosity of the first solution 240 decreases with the addition of the polymer feed 2 10 to increase the pores of the prepared biological substrate. When the polymer feed 2 10 is chitosan and its derivatives, the weight content range is about 0.5 wt% to 6 wt% of the first solution 240, and the preferred weight content range is about It is 1 wt% to 3 wt% of the first solution 240 described above. Then, a freezing process 245 is performed at a specific temperature to solidify the first solution 240 and form a solid structure 250 having a polymer-rich phase substance and a polymer-lean phase substance. Among them, the polymer The lean phase can form the pore portion of the biological substrate, while the polymer rich phase can form the pore wall portion of the biological substrate. Moreover, the pores of the biological substrate made by ′ decrease with the adjustment of the specific temperature. Secondly, a second solution 2 3 0 is provided in parallel. 20 1233943 A temperature adjustment program 260 is performed to adjust the temperature of the second solution 230 to cold; a specific temperature of the East program 245, wherein the first solution 240 is an acidic solution. And the second solution 230 is an alkaline solution; or the first solution 240 described above is an alkaline solution, and the second solution 230 is an acidic solution. When the above-mentioned polymer feed 210 is chitosan and other organisms, the first solution 240 is an acidic solution, and the first solution Luo 230 is an experimental solution. Wherein, the concentration range of the test substance contained in the second solution 2 30 is from 0.5N to 3N. Next, an acid neutralization procedure 255 is performed, and the high-molecular-rich phase is solidified by the second solution 230 described above, thereby forming a biological substrate 2 6 5. In this embodiment, before the freezing process 245, it further includes performing a defoaming process to remove air bubbles contained in the first solution 240. In addition, the above-mentioned freezing process 2 4 5 further includes a pre-drying process. The pre-drying process is performed at the above-mentioned specific temperature to vaporize the polymer-lean phase substance on the surface of the solid structure 250. On the other hand, in the temperature adjustment program 260, the above-mentioned second solution 230 can remain liquid at a specific temperature, or the second solution 230 further includes an antifreeze agent so that the second solution 230 can be at a specific temperature. The antifreeze agent further includes an ethanol component, and the ethanol concentration range is 20 wt% to 70 wt% of the second solution 230. Referring to the third figure, in a third embodiment of the present invention, the first 212133943 first provides a 鬲 molecule push Μ 2 1 Γ >, Gan, ,,,,, 一, a

The amount of material 31 is reduced and the holes are formed. When the polymer feed 310 is chitosan and its derivatives, the viscosity of 0.5% by weight of the solution containing 1 to about 34% of the above solution to the tb solution 340 decreases with the polymer. In order to increase the 6 Wt / 〇 of the prepared biological substrate, the preferred weight content range is about 1% to 3% by weight of the above solution 3 4 0; when the polymer feed 3 1 0 is alginic acid In the case of sodium and other organisms, the weight content range is about 0.5 wt / 0 to 8 wt% of the solution 340, and the preferred weight content range is about 1 wt% to 4 wt% of the solution 340. Then, the procedure 345 is performed at a specific temperature to solidify the above solution 340, and a solid structure 3 50 ′ having a polymer rich phase substance and a polymer poor phase substance is formed. Among them, the polymer poor phase can form a biological The pore portion of the substrate, and the polymer rich phase can form the pore wall portion of the biological substrate. Moreover, the pores of the prepared biological substrate decrease as the specific temperature is lowered. It is known that a parent couple agent 3 3 0 is supplied and an adjustment temperature program 3 60 is performed to adjust the temperature of the parent couple agent 3 3 0 to a specific temperature of the freezing program 3 4 5. When the high 22 1233943 molecular feed 3 1 0 is chitosan and its derivatives, the above-mentioned crosslinking agent 330 includes one of the following groups: sulfuric acid solution, aldehyde solution, dialdehyde Dialdehyde and Genipin

> Valley liquid, when the South Molecular Feed 3 10 is sodium alginate and its derivatives, the above-mentioned cross-linking agent 330 is a gasified calcium solution, and the concentration of the gasified liquid contained in the calcium chloride solution The range is from 1% to 20% by weight. Next, a gelation process 3 5 5 is performed, and the polymer-rich phase substance is gelatinized with the cross-linking agent 3 3 0 described above, and a biological substrate 365 is formed. In this embodiment, before the freezing process 345, a defoaming process is further performed to remove the air bubbles contained in the solution 34. In addition, the above-mentioned Lengkang Cheng 彳 345 also includes a pre-drying process. The pre-dried aromatic sequence is performed at the above-mentioned specific temperature to vaporize the polymer-depleted phase substance in the solid surface of 35 °. On the other hand, force adjusts temperature |

f 360 towel 'The above-mentioned cross-linking agent 33 can still maintain the state at a specific temperature' or the cross-linking agent 330 further contains a primary antibody; the east agent so that the cross-linking agent 33 can remain liquid at a specific temperature, # 中The above-mentioned resistance; the east agent further includes an ethanol component ', and the ethanol concentration range is from 20 to 70 wt% of the crosslinked Qi 33. In the above-mentioned embodiments of the present invention, the present invention discloses a method for forming a biological substrate. In response to different high scores, depending on the material or application requirements, different types of fixatives are provided under low temperature conditions to perform the fixation procedure. . Since 23 1233943, the polymer-depleted phase or the fixed polymer-rich phase is removed at a low temperature, so the polymer-rich phase structure can be maintained in the cold beam, thereby forming: a biological substrate. Moreover, the present invention is not limited to the type of solvent used, for example, a solvent having a high boiling point or a solvent that cannot be processed by the conventional cold heading drying method can be used. As the choice of solvents used increases, so does the choice of polymer materials, so bio-based materials with different materials can be made.

In summary, the present invention discloses a method for forming a biological substrate. The method has simple steps and does not require the use of a special cold beam drying device. Therefore, the present invention can meet economic benefits and industrial applicability. The above-mentioned method for forming a biological substrate includes the following steps:-dissolution process,-temperature adjustment procedure,-cold; east procedure and _ fixed procedure, the fixed procedure further includes-solid-liquid exchange procedure,-acid test neutralization procedure and Gelatinization procedure. Obviously, according to the description in the above embodiment, the present invention may have many modifications and differences. Therefore, it needs to be understood within the scope of the appended claims. In addition to the above detailed description, the present invention can be widely implemented in other embodiments. The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the patents of the present invention. All other equivalent changes or modifications made without departing from the spirit disclosed by the present invention should be included in the following applications Within the scope of the patent. [Brief description of the figure] 24 1233943 The first picture shows a 钿 4 go, middle, middle, and… according to one of the present invention is the first best, with a solid-liquid exchange 裎 bed ▲ ~ Example, spear order creature The process flow chart of the substrate; the figure shows a process flow of a biological substrate with a second acid-neutralization neutralization procedure according to one of the present invention: Figure :: The third figure shows A third preferred embodiment of the invention is a process flow chart of a biological substrate having a gelling process. Representative symbols of main parts] Polymer feed 110 Dissolving solution 1 2 0 Non-solvent 130 Dissolving process 1 3 5 Solution with specific viscosity 1 4 〇 Freezing procedure 1 4 5 Solid state structure 1 5 0 Solid-liquid exchange procedure 1 5 5 Adjustment Temperature program 1 6 〇 Biological substrate 1 6 5 Polymer feed 210 Dissolving solution 2 2 0 Second solution 2 3 0 Dissolving process 2 3 5 First solution 2 4 0 Freezing procedure 2 4 5 Solid state structure 2 5 0 Acid-base Neutralization procedure 2 5 5 Temperature adjustment procedure 2 6 0 Biological substrate 2 6 5 Polymer feed 3 1 0 Dissolving solution 320 Crosslinking agent 3 3 0 Dissolution process 3 3 5 Solution with specific viscosity 340 Freezing procedure 345 Solid state structure 3 5 0 Gelatinization program 3 5 5 Temperature adjustment program 360 Biological substrate 3 6 5

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Claims (1)

1233943 bubble, where ‘the defoaming procedure is performed before the freezing procedure. 4 · The method for forming a biological substrate as described in item 1 of the scope of the patent application, wherein the pores of the biological substrate on the dish decrease as the specific temperature is lowered. 5 · The method for forming a biological substrate as described in item 1 of the scope of the patent application, wherein the above-mentioned non-solvent can remain liquid at the specific temperature. _ 6 The method for forming a biological substrate as described in item 1 of the scope of the patent application, wherein the non-solvent further includes an antifreeze so that the non-solvent can maintain a liquid state at the specific temperature. 7. The method for forming a biological substrate according to item 1 of the scope of the patent application, wherein the above-mentioned polymer feed system is polylactic acid (PLA), polylactic acid-glycolic acid (plga), PHA Lu [P〇ly (hydroxyalkonate) s], PHB [poly (3-hydroxybutyrate)], polycaprolactone (PCL) and one of its members. 8. The method for forming a biological substrate as described in item 7 of the patent scope of Shenyan, wherein when the above polymer feed is polylactic acid (PLA) and its derivatives, the non-solvent is an aqueous ethanol solution. The ethanol concentration range of the ethanol aqueous solution is 60wn to 100wt%. 9. The method for forming a biological substrate as described in item 8 of the scope of the patent application, wherein the ethanol 27 3943 acriline range is 75 wt% to 85 wt%. The method for forming a biological substrate as described in item 7 of the Guangshenjing patent range, wherein the above-mentioned apricot (PLGA) 5 50wt% ~ 'wherein the ethanol concentration range of the ethanol aqueous solution is 2 to U. As stated The method for forming the biological substrate described in Item 1Q, wherein the above ethanol concentration range is 25 wt% to 35 wt%. 12. A method for forming a biological substrate, the method comprising: providing a polymer feed and a dissolving solution; dissolving the polymer feed with the dissolving solution to form a first solution having a specific viscosity At one, a freezing process is performed at a specific temperature to solidify the solution and form a solid structure having a polymer-rich phase substance and a polymer-lean phase substance, wherein the polymer-lean phase can form pores of a biological substrate. Seven points, and the polymer rich phase can form the pore wall portion of the biological substrate; under the specific degree of fermentation, a pre-drying process is performed to vaporize the polymer poor phase substance on the surface of the solid structure; i. The first solution and a temperature adjustment program are performed to adjust the j / mixed liquid to the specific temperature of the freezing program; an acid test and neutralization program are performed with the second solution to make the polymer rich phase solidify 28 1233943 This does not form a biological substrate. 13. The method for forming a biological substrate as described in item 12 of the scope of the patent application, wherein the viscosity of the above-mentioned solution decreases as the addition amount of the polymer feed decreases to increase the prepared biological substrate. Holes. W · The method for forming a biological substrate as described in item 12 of the scope of the patent application, further comprising a defoaming process to remove air bubbles contained in the first solution, wherein the defoaming process is performed before the freezing process . 15. According to the alkaline method of the biological button described in Article 12, the pores of the biological substrate described above decrease as the specific temperature is lowered. 16. The method for forming a biological substrate according to item 12 of the scope of the patent application, wherein the second solution described above can still maintain a liquid state at the specific temperature. 17. The method for forming a biological substrate as described in item 12 of the patent scope of Shenyan, wherein the first solution described above further comprises -anti; an agent so that the second solution can remain liquid at the specific temperature. 0 18 · For example, the nth refrigerant in the patent application range further contains an ethanol component to 70% by weight. The method for forming a biological substrate as described in the above item, wherein the above-mentioned anti- 'and the ethanol concentration range is 20% by weight of the second solution 29 1233943 19 · The method for forming the biological substrate as described in item 12 of the scope of patent application Among them, the valley solution is an acidic solution, and the second solution is an alkaline solution. 20_ The method for forming a biological substrate according to item 12 of the patent scope of Shenyan, wherein the first solution is an alkaline solution, and the second solution is an acidic solution. 21. The method for forming a biological substrate according to item 19 in the scope of the patent application, wherein the above-mentioned high molecular feed is chitosan and its derivative. _ 22. The method for forming a biological substrate as described in item 21 of the scope of patent application, wherein the concentration range of the alkaline substance contained in the second solution is 0.5N to 3N. 23. A method for forming a biological substrate, the method for forming a biological substrate comprising: providing a polymer feed and a dissolving solution; dissolving the polymer with the dissolving solution to form a solution having a specific viscosity; A freezing process is performed at a specific temperature to solidify the solution 'and form a solid structure having a polymer-rich phase substance and a polymer-lean phase substance, wherein the polymer-lean phase can form a pore portion of a biological substrate, and the polymer The rich phase can form the pore wall portion of the biological substrate; provide a cross-linking agent and perform a temperature adjustment program to adjust the freezing temperature of the-parent cross-linking agent to the freezing process; and-gelatinize by the cross-linking agent The procedure is to gel the polymer 30 1233943 phase-rich material and form a biological substrate. 24. The method for forming a biological substrate as described in item 23 of the scope of the patent application, wherein the viscosity of the above-mentioned solution decreases as the addition amount of the polymer feed decreases to increase the prepared biological substrate Holes. 25. The method for forming a biological substrate as described in item 23 of the scope of the patent application, further comprising a defoaming procedure to remove the bubbles contained in the solution, wherein the defoaming procedure is performed before the freezing procedure. 26. The method for forming a biological substrate as described in item 23 of the scope of patent application, wherein the pores of the biological substrate are reduced as the specific temperature is lowered. 27. The method for forming a biological substrate according to item 23 of the scope of the patent application, wherein the freezing process further includes a pre-drying process, the pre-drying process is performed at the specific temperature to vaporize the surface of the solid structure The polymer has a poor phase substance. 28. The method for forming a biological substrate as described in item 23 of the scope of patent application, wherein the above-mentioned cross-linking agent can remain liquid at the specific temperature 1233943. 29. In the forming method, the above-mentioned cross-linking agent further includes an antifreeze agent so that the non-solvent can maintain a liquid state at the specific temperature. 30. The method for forming a biological substrate according to item 29 of the scope of the patent application, wherein the above-mentioned anti / east agent further comprises an ethanol component, and the ethanol concentration range is 20 wt% to 70 wt% of the cross-linking agent. 31. The method for forming a biological substrate according to item 23 of the patent application, wherein the above-mentioned high molecular feed is chitosan and its derivative. 32. The method for forming a biological substrate according to item 23 of the scope of the patent application, wherein the above-mentioned high molecular feed is alginate and its derivatives.