JP2012082244A - Method for manufacturing silk fibroin porous body - Google Patents
Method for manufacturing silk fibroin porous body Download PDFInfo
- Publication number
- JP2012082244A JP2012082244A JP2010227030A JP2010227030A JP2012082244A JP 2012082244 A JP2012082244 A JP 2012082244A JP 2010227030 A JP2010227030 A JP 2010227030A JP 2010227030 A JP2010227030 A JP 2010227030A JP 2012082244 A JP2012082244 A JP 2012082244A
- Authority
- JP
- Japan
- Prior art keywords
- silk fibroin
- acid
- porous material
- solution
- porous body
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 144
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title abstract description 23
- 239000000243 solution Substances 0.000 claims abstract description 41
- 150000007933 aliphatic carboxylic acids Chemical class 0.000 claims abstract description 30
- 239000007864 aqueous solution Substances 0.000 claims abstract description 28
- 230000008014 freezing Effects 0.000 claims abstract description 22
- 238000007710 freezing Methods 0.000 claims abstract description 22
- 238000010257 thawing Methods 0.000 claims abstract description 7
- 239000011148 porous material Substances 0.000 claims description 79
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 235000011054 acetic acid Nutrition 0.000 claims description 5
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 4
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 4
- PVEOYINWKBTPIZ-UHFFFAOYSA-N but-3-enoic acid Chemical compound OC(=O)CC=C PVEOYINWKBTPIZ-UHFFFAOYSA-N 0.000 claims description 4
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 4
- 230000008018 melting Effects 0.000 claims description 4
- 238000002844 melting Methods 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- LDHQCZJRKDOVOX-NSCUHMNNSA-N crotonic acid Chemical compound C\C=C\C(O)=O LDHQCZJRKDOVOX-NSCUHMNNSA-N 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 239000004310 lactic acid Substances 0.000 claims description 2
- 235000014655 lactic acid Nutrition 0.000 claims description 2
- 150000002763 monocarboxylic acids Chemical class 0.000 claims description 2
- 235000019260 propionic acid Nutrition 0.000 claims description 2
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 150000003628 tricarboxylic acids Chemical class 0.000 claims description 2
- 239000001384 succinic acid Substances 0.000 claims 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 238000001878 scanning electron micrograph Methods 0.000 description 10
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 239000000654 additive Substances 0.000 description 6
- 230000000996 additive effect Effects 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 125000004432 carbon atom Chemical group C* 0.000 description 4
- -1 cellulose and chitin Chemical group 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 238000001879 gelation Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108010076876 Keratins Proteins 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 210000003746 feather Anatomy 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920000139 polyethylene terephthalate Polymers 0.000 description 2
- 239000005020 polyethylene terephthalate Substances 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- BFKJFAAPBSQJPD-UHFFFAOYSA-N tetrafluoroethene Chemical group FC(F)=C(F)F BFKJFAAPBSQJPD-UHFFFAOYSA-N 0.000 description 2
- 230000017423 tissue regeneration Effects 0.000 description 2
- 210000002268 wool Anatomy 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000255794 Bombyx mandarina Species 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000001991 dicarboxylic acids Chemical class 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012520 frozen sample Substances 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 150000002762 monocarboxylic acid derivatives Chemical class 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)
Abstract
Description
本発明は、シルクフィブロイン多孔質体の製造方法に関する。 The present invention relates to a method for producing a silk fibroin porous material.
タンパク質や糖類などの生物由来物質を利用して作製可能である多孔質体は、医療分野、生活日用品分野、浄水分野、化粧品・エステ分野、組織工学や再生医工学における細胞培養支持体や組織再生支持体など産業上幅広い分野で利用される。
これら多孔質体を構成する生体由来物質としては、セルロースやキチン等の糖類、コラーゲン、ケラチン、シルクフィブロイン等のタンパク質群が知られている。
Porous materials that can be produced using biological materials such as proteins and saccharides are cell culture supports and tissue regeneration in the medical field, daily necessities field, water purification field, cosmetics / esthetic field, tissue engineering and regenerative medical engineering. Used in a wide range of industrial fields such as supports.
As biologically-derived substances constituting these porous bodies, saccharides such as cellulose and chitin, and protein groups such as collagen, keratin, and silk fibroin are known.
このうち、タンパク質としては、コラーゲンが一番よく利用されてきたが、ＢＳＥ問題が発生してから牛由来のコラーゲンを利用することが非常に難しくなってきた。また、ケラチンは、羊毛や羽毛から得ることができるが、原料入手に問題があり、工業的に利用することは難しい。羊毛は、原料価格が非常に高騰しており、羽毛に関しては市場がないため、原料を入手することができない。これらに対して、シルクフィブロインは、原料入手の観点からは、安定に供給されることが期待でき、さらに価格も安定しているので、工業的に利用することが容易である。
シルクフィブロインは、衣類用途以外に、手術用縫合糸として長く使用されてきた実績があり、現在では食品や化粧品の添加物としても利用され、人体に対する安全性にも問題がないことから上記した多孔質体の利用分野に十分利用可能である。
Of these, collagen has been most often used as a protein, but it has become very difficult to utilize bovine-derived collagen after the occurrence of the BSE problem. Keratin can be obtained from wool and feathers, but there is a problem in obtaining raw materials and it is difficult to use it industrially. For wool, the raw material price is soaring and there is no market for feathers, so it is not possible to obtain the raw material. On the other hand, silk fibroin can be expected to be stably supplied from the viewpoint of obtaining raw materials, and the price is also stable, so that it is easy to use industrially.
Silk fibroin has a long track record of being used as a surgical suture for purposes other than clothing, and is currently used as an additive in foods and cosmetics. It is fully usable in the field of use of masses.
シルクフィブロイン多孔質体を作製する手法に関しては、いくつか報告がある。例えば、シルクフィブロイン水溶液を急速冷凍したのち結晶化溶媒に浸漬し、融解と結晶化を同時進行することによって得る方法がある（特許文献１）。しかしながら、この方法は結晶化溶媒である有機溶媒を大量に使用する必要があり、さらに溶媒の残留の可能性も否定できず、医療分野等の上記した応用分野での使用には問題がある。次に、シルクフィブロイン水溶液のｐＨを６以下に保持してゲル化させるか又はその水溶液に貧溶媒を添加してゲル化させ、得られたゲルを凍結乾燥して多孔質体を作製する方法がある（特許文献２）。しかしながら、この方法は十分な強度をもった多孔質体を得ることはできない。他に、シルクフィブロイン水溶液を冷凍した後に長時間凍結状態を維持することで多孔質体を作製する手法が報告されている（特許文献３）。しかしながら、発明者らの検討ではこの手法は再現性が乏しく、多孔質体が作製できないことが多い。
上記したシルクフィブロイン多孔質体の作製手法と比較すると、確実で簡便な手法が報告されている（特許文献４；非特許文献１）。シルクフィブロイン水溶液に対して少量の水溶性有機溶媒を添加した後に、一定時間冷凍して融解することによってシルクフィブロイン多孔質体が得られる手法である。この手法では、少量使用した有機溶媒も超純水による洗浄工程による除去から残留有機溶媒もほぼなくなり、さらには、得られた含水状態の多孔質体は、それまでに報告されていたものよりも強度が高く形態安定性に優れている。
There are several reports on the technique for producing silk fibroin porous materials. For example, there is a method in which a silk fibroin aqueous solution is rapidly frozen and then immersed in a crystallization solvent to simultaneously melt and crystallize (Patent Document 1). However, this method requires the use of a large amount of an organic solvent, which is a crystallization solvent. Further, the possibility of remaining of the solvent cannot be denied, and there is a problem in use in the above-mentioned application fields such as the medical field. Next, there is a method for producing a porous body by maintaining the pH of the silk fibroin aqueous solution at 6 or less to cause gelation, or by adding a poor solvent to the aqueous solution for gelation, and freeze-drying the obtained gel. Yes (Patent Document 2). However, this method cannot obtain a porous body having sufficient strength. In addition, a method for producing a porous body by maintaining a frozen state for a long time after freezing a silk fibroin aqueous solution has been reported (Patent Document 3). However, according to the studies by the inventors, this method is poor in reproducibility, and it is often impossible to produce a porous body.
Compared with the above-described method for producing a silk fibroin porous material, a reliable and simple method has been reported (Patent Document 4; Non-Patent Document 1). This is a technique in which a silk fibroin porous material is obtained by adding a small amount of a water-soluble organic solvent to a silk fibroin aqueous solution and then freezing and melting for a certain time. In this method, the organic solvent used in a small amount and the residual organic solvent are almost eliminated from the removal by the washing process with ultrapure water. High strength and excellent shape stability.
上記の生物由来物質から得られる多孔質体の利用可能分野あるいは使用方法は幅広く、各用途に応じて要求される特性、例えば多孔質体の強度などの特性は多岐にわたっている。とりわけ、より高い強度を有するものに対する要求が増加しており、従来の技術を用いて得られるシルクフィブロイン多孔質体の製造方法では対応しきれない場合が増える傾向にある。
そこで、本発明は、多孔質体の強度を容易に調整し、より高い強度を有する多孔質体を製造する方法を提供することを目的とする。
There are a wide range of fields and methods of use of porous bodies obtained from the above-mentioned biological materials, and properties required for each application, for example, properties such as the strength of the porous body are diverse. In particular, there is an increasing demand for a material having higher strength, and there is a tendency that the number of cases in which the method for producing a silk fibroin porous material obtained by using a conventional technique cannot be handled is increased.
Then, this invention aims at providing the method of adjusting the intensity | strength of a porous body easily and manufacturing the porous body which has higher intensity | strength.
本発明者は、前記課題を達成するために鋭意研究を重ねた結果、下記の発明により当該課題を解決できることを見出した。すなわち本発明は、シルクフィブロインの含有量が１０〜５０質量％であるシルクフィブロイン水溶液に脂肪族カルボン酸を添加したシルクフィブロイン溶液を凍結し、融解することを特徴とするシルクフィブロイン多孔質体の製造方法を提供するものである。 As a result of intensive studies to achieve the above problems, the present inventor has found that the problems can be solved by the following invention. That is, the present invention provides a silk fibroin porous material characterized by freezing and thawing a silk fibroin solution obtained by adding an aliphatic carboxylic acid to a silk fibroin aqueous solution having a silk fibroin content of 10 to 50% by mass. A method is provided.
本発明によれば、様々な多孔質体の強度を有するシルクフィブロイン多孔質体、とりわけより高い強度を有するシルクフィブロイン多孔質体を簡便に得ることができる。 ADVANTAGE OF THE INVENTION According to this invention, the silk fibroin porous body which has the intensity | strength of various porous bodies, especially the silk fibroin porous body which has higher intensity | strength can be obtained simply.
本発明のシルクフィブロイン多孔質体の製造方法は、シルクフィブロインの含有量が１０〜５０質量％であるシルクフィブロイン水溶液に脂肪族カルボン酸を添加したシルクフィブロイン溶液を凍結し、融解することを特徴とする。
また、本発明の製造方法においては、シルクフィブロイン溶液を該溶液が凝固しない温度で１時間以上静置した後、凍結し、融解することが好ましく、また、融解後に得られた多孔質体を純水中に浸漬して前記脂肪族カルボン酸を除去する工程をさらに有することが好ましい。
The method for producing a silk fibroin porous material of the present invention is characterized by freezing and thawing a silk fibroin solution obtained by adding an aliphatic carboxylic acid to a silk fibroin aqueous solution having a silk fibroin content of 10 to 50% by mass. To do.
In the production method of the present invention, it is preferable that the silk fibroin solution is allowed to stand for 1 hour or more at a temperature at which the solution does not solidify, and then frozen and thawed. It is preferable to further include a step of removing the aliphatic carboxylic acid by immersing in water.
本発明で用いられるシルクフィブロインは、家蚕、野蚕、天蚕等の蚕から産生されるものであればいずれでもよく、その製造方法も問わない。本発明では、シルクフィブロイン水溶液として用いるが、シルクフィブロインは溶解性が悪く、直接水には溶解することが困難である。シルクフィブロイン水溶液を得る方法としては、公知のいかなる手法を用いてもよいが、高濃度の臭化リチウム水溶液にシルクフィブロインを溶解後、透析による脱塩、風乾による濃縮を経る手法が簡便である。ここで、臭化リチウム水溶液の濃度は、８〜１０Ｍが好ましく、８．５〜９．５Ｍがより好ましい。臭化リチウム水溶液の濃度が、上記範囲内であると、シルクフィブロイン溶液がゲル化しにくく、安定して均一な構造のシルクフィブロイン多孔質体を得ることができる。 The silk fibroin used in the present invention may be any silk fibroin produced from silkworms such as rabbits, wild silkworms, and tengu, and the manufacturing method thereof is not limited. In the present invention, silk fibroin is used as an aqueous solution of silk fibroin, but silk fibroin is poorly soluble and difficult to dissolve directly in water. As a method for obtaining a silk fibroin aqueous solution, any known method may be used, but a method in which silk fibroin is dissolved in a high concentration lithium bromide aqueous solution, followed by desalting by dialysis and concentration by air drying is simple. Here, the concentration of the lithium bromide aqueous solution is preferably 8 to 10M, and more preferably 8.5 to 9.5M. When the concentration of the lithium bromide aqueous solution is within the above range, the silk fibroin solution is difficult to gel, and a silk fibroin porous body having a stable and uniform structure can be obtained.
本発明のシルクフィブロイン多孔質体の製造方法において、シルクフィブロイン水溶液中のシルクフィブロインの濃度は、脂肪族カルボン酸を添加したシルクフィブロイン溶液中で１０〜５０質量％となるような濃度であることを要する。この範囲内に設定することで、高い強度を持ったシルクフィブロイン多孔質体を効率的に製造することができる。また、フィブロインの濃度を調節することで、必要に応じた強度のシルクフィブロイン多孔質体を得ることができる。例えば、より強度が高い多孔質体が必要であればフィブロイン濃度を３０〜５０質量％とすることが好ましく、４０〜５０質量％とすることがより好ましい。 In the method for producing a silk fibroin porous material of the present invention, the concentration of silk fibroin in the silk fibroin aqueous solution is such that the concentration is 10 to 50% by mass in the silk fibroin solution to which an aliphatic carboxylic acid is added. Cost. By setting within this range, a silk fibroin porous body having high strength can be efficiently produced. Further, by adjusting the fibroin concentration, a silk fibroin porous material having a strength as required can be obtained. For example, if a porous body having higher strength is required, the fibroin concentration is preferably 30 to 50% by mass, and more preferably 40 to 50% by mass.
本発明において、シルクフィブロイン水溶液に添加する脂肪族カルボン酸は、シルクフィブロインの多孔質化を促進させる効果を有する添加剤である。脂肪族カルボン酸としては、特に制限はないが、水溶性のものが好ましく、水への溶解度が高いものがより好ましい。また、本発明において用いられる脂肪族カルボン酸としては、ｐＫａが、５．０以下のものが好ましく、３．０〜５．０のものがより好ましく、３．５〜５．０のものがさらに好ましい。
本発明において用いられる脂肪族カルボン酸としては、例えば、好ましくは炭素数１〜６、より好ましくは炭素数３〜５の飽和または不飽和のモノカルボン酸、ジカルボン酸、トリカルボン酸を好ましく用いることができ、例えば、蟻酸、酢酸、プロピオン酸、酪酸、乳酸、アクリル酸、２−ブテン酸、３−ブテン酸などのモノカルボン酸；シュウ酸、マロン酸、コハク酸、フマル酸などのジカルボン酸などが好ましく挙げられる。これらの脂肪族カルボン酸は、単独あるいは２種以上組み合わせて使用することができる。
In the present invention, the aliphatic carboxylic acid added to the silk fibroin aqueous solution is an additive having an effect of promoting the porous formation of silk fibroin. The aliphatic carboxylic acid is not particularly limited, but is preferably water-soluble and more preferably has high solubility in water. The aliphatic carboxylic acid used in the present invention preferably has a pKa of 5.0 or less, more preferably 3.0 to 5.0, and even more preferably 3.5 to 5.0. preferable.
As the aliphatic carboxylic acid used in the present invention, for example, a saturated or unsaturated monocarboxylic acid, dicarboxylic acid or tricarboxylic acid having preferably 1 to 6 carbon atoms, more preferably 3 to 5 carbon atoms is preferably used. For example, monocarboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, lactic acid, acrylic acid, 2-butenoic acid, and 3-butenoic acid; dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, and fumaric acid Preferably mentioned. These aliphatic carboxylic acids can be used alone or in combination of two or more.
本発明において、高い強度を有するシルクフィブロイン多孔質体を得ようとする場合、直鎖の構造を有し、炭素数が多い脂肪族カルボン酸を用いることが好ましい。また、本発明において、小さい細孔直径を有するシルクフィブロイン多孔質体を得ようとする場合は、脂肪族カルボン酸を使用することが好ましく、より小さい細孔直径のものを得ようとするには、直鎖の構造を有し、炭素数が多い脂肪族カルボン酸を用いることが好ましい。 In the present invention, when obtaining a silk fibroin porous material having high strength, it is preferable to use an aliphatic carboxylic acid having a linear structure and having a large number of carbon atoms. In the present invention, when a silk fibroin porous material having a small pore diameter is to be obtained, it is preferable to use an aliphatic carboxylic acid, and to obtain a material having a smaller pore diameter. It is preferable to use an aliphatic carboxylic acid having a linear structure and a large number of carbon atoms.
シルクフィブロイン水溶液に添加する脂肪族カルボン酸の量は、シルクフィブロイン溶液中で０．０１〜１８質量％であることが好ましく、０．１〜５質量％であることがより好ましく、０．５〜４質量％であることがさらに好ましい。この範囲内に設定することで、高い強度を持ったシルクフィブロイン多孔質体を製造することができる。また、４質量％以下であれば、シルクフィブロイン水溶液に脂肪族カルボン酸を添加したシルクフィブロイン溶液を静置する際、該溶液がゲル化しにくく、安定して均一な構造のシルクフィブロイン多孔質体が得られる。また、シルクフィブロイン水溶液に脂肪族カルボン酸を添加する際に起こりやすいゲル化を予防するために、目的とするフィブロイン濃度よりも高濃度なシルクフィブロイン水溶液を予め調製しておき、そこに脂肪族カルボン酸の希釈水溶液を加えることがさらに好ましい。 The amount of the aliphatic carboxylic acid added to the silk fibroin aqueous solution is preferably 0.01 to 18% by mass, more preferably 0.1 to 5% by mass in the silk fibroin solution, and 0.5 to More preferably, it is 4 mass%. By setting within this range, a silk fibroin porous body having high strength can be produced. In addition, if it is 4% by mass or less, when the silk fibroin solution obtained by adding an aliphatic carboxylic acid to the silk fibroin aqueous solution is allowed to stand, the silk fibroin porous body having a stable and uniform structure is difficult to gel. can get. In addition, in order to prevent gelation that is likely to occur when an aliphatic carboxylic acid is added to a silk fibroin aqueous solution, a silk fibroin aqueous solution having a concentration higher than the target fibroin concentration is prepared in advance, and an aliphatic carboxylic acid is added thereto. More preferably, a dilute aqueous solution of acid is added.
本発明の製造方法では、シルクフィブロイン水溶液に脂肪族カルボン酸を添加したシルクフィブロイン溶液を、該溶液が凝固しない温度で１０時間以上静置することが好ましい。静置の工程を加えることにより、高い強度を有し、かつ安定して均一な構造のシルクフィブロイン多孔質体が得られる。該シルクフィブロイン溶液の静置は、該溶液を型あるいは容器に流し込んで、所定の温度条件下で行えばよい。
静置する際の温度は、凝固しない温度であれば特に制限はないが、凝固のしにくさ、溶液のゲル化のしにくさ、あるいはフィブロイン分子の分解の起こりにくさを考慮すると、−５〜５０℃であることが好ましく、−３〜２０℃がより好ましく、３〜１０℃がさらに好ましい。シルクフィブロイン溶液を静置する温度を調節することで、得られるシルクフィブロイン多孔質体の細孔直径や強度を調整することができ、温度を３〜１０℃とすることで、特に細孔直径が小さく、強度の高い多孔質体が得られる。
In the production method of the present invention, it is preferable to leave a silk fibroin solution obtained by adding an aliphatic carboxylic acid to a silk fibroin aqueous solution at a temperature at which the solution does not solidify for 10 hours or more. By adding the stationary step, a silk fibroin porous body having high strength and a stable and uniform structure can be obtained. The silk fibroin solution may be left standing under a predetermined temperature condition by pouring the solution into a mold or a container.
The temperature at the time of standing is not particularly limited as long as it does not coagulate, but it is −5 in consideration of difficulty of coagulation, difficulty of gelation of solution, or difficulty of decomposition of fibroin molecules. It is preferable that it is -50 degreeC, -3-20 degreeC is more preferable, and 3-10 degreeC is further more preferable. By adjusting the temperature at which the silk fibroin solution is allowed to stand, the pore diameter and strength of the resulting silk fibroin porous material can be adjusted. A small porous body having high strength can be obtained.
シルクフィブロイン溶液を静置する時間は、１０時間以上であれば特に制限はないが、製造効率を考慮すると、３００時間以内が好ましく、２００時間以内がより好ましい。シルクフィブロイン溶液を静置する時間を調節することで、得られるシルクフィブロイン多孔質体の細孔直径や強度を調節することができ、時間を長くすると、細孔直径が小さく、より強度が高い多孔質体が得られる。 The time for allowing the silk fibroin solution to stand is not particularly limited as long as it is 10 hours or longer, but considering production efficiency, it is preferably within 300 hours, and more preferably within 200 hours. By adjusting the time for which the silk fibroin solution is allowed to stand, the pore diameter and strength of the resulting silk fibroin porous material can be adjusted. If the time is increased, the pore diameter is reduced and the porosity is higher. A mass is obtained.
本発明の製造方法では、シルクフィブロイン水溶液と脂肪族カルボン酸とを混合してシルクフィブロイン溶液を調製した後、あるいは該シルクフィブロイン溶液を調製して、該シルクフィブロイン溶液を静置した後、低温恒温槽中に入れて凍結させ、次いで融解することによって、シルクフィブロイン多孔質体を製造する。
凍結温度は、脂肪族カルボン酸を含有させたシルクフィブロイン溶液が凍結する温度であれば特に制限されないが、−４〜−４０℃が好ましく、−１０〜−３０℃がより好ましく、−１０〜−２０℃がさらに好ましい。凍結温度を上記範囲内にすると、シルクフィブロイン多孔質体を再現よく得ることができる。
凍結時間は、十分に凍結し、かつ凍結状態を一定時間保持できるよう、所定の凍結温度で２時間以上であることが好ましく、４時間以上がより好ましい。
In the production method of the present invention, after preparing a silk fibroin solution by mixing an aqueous silk fibroin solution and an aliphatic carboxylic acid, or after preparing the silk fibroin solution and allowing the silk fibroin solution to stand, A silk fibroin porous material is produced by freezing in a bath and then thawing.
The freezing temperature is not particularly limited as long as the silk fibroin solution containing the aliphatic carboxylic acid is frozen, but is preferably −4 to −40 ° C., more preferably −10 to −30 ° C., and more preferably −10 to − 20 ° C. is more preferable. When the freezing temperature is within the above range, a silk fibroin porous material can be obtained with good reproducibility.
The freezing time is preferably 2 hours or more, more preferably 4 hours or more at a predetermined freezing temperature so that it can be sufficiently frozen and kept in a frozen state for a certain period of time.
凍結の方法としては、静置後のシルクフィブロイン溶液を一気に凍結温度まで下げて凍結してもよいが、静置を常温程度の温度条件で行った場合には、静置の後、凍結の前に過冷却状態を経て凍結することが好ましい。なお、溶液が過冷却状態となるような温度条件で静置を行った場合には、改めて過冷却状態を経て凍結しなくてもよい。
過冷却状態とする方法としては、例えば一旦、５〜−１０℃程度、好ましくは０〜−５℃程度で、３０分以上保持して容器内を均一にして過冷却状態としてから、凍結温度まで下げて凍結することで、均一な構造のシルクフィブロイン多孔質体を得ることができる。静置あるいは過冷却状態から凍結温度までにかける時間を調整することで、多孔質体の構造や強度をある程度制御することが可能であり、例えば、０．１〜２℃／ｈ程度でゆっくり温度を下げると、より高い強度のシルクフィブロイン多孔質体が得られる。
As a freezing method, the silk fibroin solution after standing may be frozen at a freezing temperature all at once. However, when the standing is performed at room temperature, the standing silk fibroin solution may be frozen after standing and before freezing. It is preferable to freeze through a supercooled state. In addition, when it leaves still by the temperature conditions that a solution will be in a supercooled state, it does not need to freeze through a supercooled state again.
As a method for making the supercooled state, for example, once at about 5 to −10 ° C., preferably about 0 to −5 ° C., hold it for 30 minutes or more to make the inside of the container uniform, and then to the freezing temperature. By lowering and freezing, a silk fibroin porous body having a uniform structure can be obtained. It is possible to control the structure and strength of the porous body to some extent by adjusting the time taken from the standing or supercooled state to the freezing temperature. For example, the temperature is slowly increased to about 0.1 to 2 ° C./h. When the value is lowered, a silk fibroin porous body having higher strength can be obtained.
上記のようにして凍結した後、凍結したシルクフィブロイン溶液を、融解することによってシルクフィブロイン多孔質体が得られる。融解の方法は特に制限はないが、自然融解のほか、恒温槽内に保管する方法などが挙げられる。 After freezing as described above, a silk fibroin porous material is obtained by thawing the frozen silk fibroin solution. The melting method is not particularly limited, and examples include natural melting and a method of storing in a thermostatic bath.
得られたシルクフィブロイン多孔質体には脂肪族カルボン酸が含まれるが、用途に応じて、脂肪族カルボン酸を除去する必要がある場合には、適当な方法でシルクフィブロイン多孔質体から脂肪族カルボン酸を除去して用いることができる。たとえば、多孔質体を、純水中に浸漬して、脂肪族カルボン酸を除去することが最も簡便な方法として挙げられる。 The obtained silk fibroin porous material contains an aliphatic carboxylic acid. If it is necessary to remove the aliphatic carboxylic acid depending on the use, the silk fibroin porous material can be removed from the silk fibroin porous material by an appropriate method. It can be used after removing the carboxylic acid. For example, the simplest method is to remove the aliphatic carboxylic acid by immersing the porous body in pure water.
本発明の製造方法により得られるシルクフィブロイン多孔質体は、多孔質体作製時の型や容器を適宜選択することにより、フィルム状、ブロック状、管状等、目的に応じた形状とすることができる。型や容器としては、シルクフィブロイン溶液が流出しない形状・形態のものであれば制限はなく、その素材としては、鉄、ステンレス、アルミニウム、金、銀、銅などの熱伝導率が高い素材を用いることが、均一な構造のシルクフィブロイン多孔質体を得る観点から好ましい。また、型や容器の厚さは、その機能と凍結の際の膨張などによる変形などを防止する観点から、０．５ｍｍ以上であることが好ましく、取り扱いが容易で、冷却効率的な観点から、より好ましくは１〜３ｍｍである。 The silk fibroin porous material obtained by the production method of the present invention can be made into a shape suitable for the purpose, such as a film shape, a block shape, a tubular shape, etc., by appropriately selecting a mold and a container for producing the porous material. . There are no restrictions on the shape or container of the silk fibroin solution as long as it does not flow out. The material used is a material with high thermal conductivity such as iron, stainless steel, aluminum, gold, silver, or copper. It is preferable from the viewpoint of obtaining a silk fibroin porous body having a uniform structure. The thickness of the mold and the container is preferably 0.5 mm or more from the viewpoint of preventing deformation due to expansion and the like of its function and freezing, from the viewpoint of easy handling and cooling efficiency, More preferably, it is 1-3 mm.
これらの型や容器から取り出したシルクフィブロイン多孔質体において、該型や容器に触れる面には、厚さ１〜１００μｍ程度のフィルム層が形成している。該フィルム層は、細孔を有さない層である。ここで、細孔を有さないとは、細孔は原則存在しない、すなわち実質的に細孔を有さず、細孔を有していてもその細孔は製造上の欠陥として生じてしまうものであることを意味する。より具体的には、細孔を有する場合でも、その細孔直径が０．５μｍ以上の細孔の個数は２０個／ｍｍ2以下、好ましくは１０個／ｍｍ2以下程度のものであり、その細孔の割合は面積比で１０％以下、好ましくは５％以下程度のものである。ここで、フィルム層の細孔の面積比、細孔の個数及び細孔直径は、走査型電子顕微鏡写真を、画像解析ソフトＩｍａｇｅＪ（アメリカ国立衛生研究所製）を用いて画像処理することで測定したものである。 In the silk fibroin porous material taken out from these molds and containers, a film layer having a thickness of about 1 to 100 μm is formed on the surface that touches the molds and containers. The film layer is a layer having no pores. Here, “having no pores” means that the pores do not exist in principle, that is, they do not substantially have pores, and even if they have pores, the pores are produced as manufacturing defects. It means to be a thing. More specifically, even when having pores, the number of pores having a pore diameter of 0.5 μm or more is 20 / mm 2 or less, preferably about 10 / mm 2 or less. The ratio of the pores is 10% or less, preferably about 5% or less in terms of area ratio. Here, the area ratio of the pores of the film layer, the number of pores and the pore diameter are measured by subjecting a scanning electron micrograph to image processing using image analysis software ImageJ (manufactured by National Institutes of Health). It is a thing.
一方、本発明の製造方法により得られるシルクフィブロイン多孔質体において、上記した多孔質層は、多数の細孔を有するスポンジ状の多孔質構造を有するものであり、好ましくは１〜３００μｍの孔径を有する。このように、該フィルム層は、細孔の観点から該フィルム層に囲まれた内側にある多孔質体とは全く異なる性状を有する層である。 On the other hand, in the silk fibroin porous material obtained by the production method of the present invention, the porous layer described above has a sponge-like porous structure having a large number of pores, and preferably has a pore diameter of 1 to 300 μm. Have. Thus, the film layer is a layer having completely different properties from the porous body inside surrounded by the film layer from the viewpoint of pores.
本発明の製造方法で得られるシルクフィブロイン多孔質体は、多孔質層、及びそのまわりを被覆するフィルム層を含む構成を有するものであり、その用途に応じて、フィルム層を取り除いて用いてもよいし、積極的にフィルム層を残して用いてもよいものである。例えば、ブロック状の型あるいは容器で作製したシルクフィブロイン多孔質体の場合、側面の四面のフィルム層を取り除き、多孔質層の部分で切削すると、多孔質層とフィルム層とを含む構成を有するシルクフィブロイン多孔質体を得ることができる（図１を参照）。ここで、フィルム層の除去や多孔質層の切削には、切削工程で常用される器具、装置などを制限なく用いることができるが、鋭利な刃物を用いることが好ましい。 The silk fibroin porous material obtained by the production method of the present invention has a structure including a porous layer and a film layer covering the porous layer, and the film layer may be removed depending on the use. It may be used positively leaving a film layer. For example, in the case of a silk fibroin porous body made of a block-shaped mold or container, the silk having a structure including a porous layer and a film layer is obtained by removing the four side film layers and cutting the porous layer portion. A fibroin porous body can be obtained (see FIG. 1). Here, for the removal of the film layer and the cutting of the porous layer, tools, devices and the like that are commonly used in the cutting process can be used without limitation, but it is preferable to use a sharp blade.
また、本発明の製造方法で用いられる型や容器は、前記フィルム層の構造や厚さを制御することを目的として、その内側のシルクフィブロイン溶液と接する内壁面に、シートを設けることができる。
該シートとしては、ポリテトラフルオロエチレン（ＰＴＦＥ）、テトラフルオロエチレン・ヘキサフルオロプロピレン共重合体（ＦＥＰ）やテトラフルオロエチレン・パーフルオロアルキルビニルエーテル共重合体（ＰＦＡ）などのフッ素樹脂からなるシート、あるいはポリエチレンテレフタレート（ＰＥＴ）やポリプロピレン（ＰＰ）などからなる離型処理されたシートなどが好ましく挙げられる。これらのシートを用いた場合、細孔が少なく平滑なフィルム層を得ることができる。また、フィルム層を設けたくない場合は、ろ紙などといった表面が粗いシートを設けることもできる。これらのシートの採用については、多孔質体の用途に応じて、適宜選択すればよい。
また、シートは、熱伝導を阻害しにくい厚さ１ｍｍ以下のものを用いることが好ましい。
Moreover, the mold | type and container used with the manufacturing method of this invention can provide a sheet | seat on the inner wall surface in contact with the silk fibroin solution inside for the purpose of controlling the structure and thickness of the said film layer.
As the sheet, a sheet made of a fluororesin such as polytetrafluoroethylene (PTFE), tetrafluoroethylene / hexafluoropropylene copolymer (FEP) or tetrafluoroethylene / perfluoroalkyl vinyl ether copolymer (PFA), or Preferable examples include release-treated sheets made of polyethylene terephthalate (PET) or polypropylene (PP). When these sheets are used, a smooth film layer with few pores can be obtained. In addition, when it is not desired to provide a film layer, a sheet having a rough surface such as filter paper can be provided. About adoption of these sheets, what is necessary is just to select suitably according to the use of a porous body.
In addition, it is preferable to use a sheet having a thickness of 1 mm or less that hardly inhibits heat conduction.
本発明の製造方法により得られるシルクフィブロイン多孔質体において、多孔質層は、上記したように、多数の細孔を有するスポンジ状の多孔質構造であり、通常この多孔質層には凍結乾燥などにより水除去を行わなければ水が含まれ、含水状態で硬い構造物である。また、多孔質体を凍結乾燥することにより、シルクフィブロイン多孔質体の乾燥品を得ることができる。 In the silk fibroin porous material obtained by the production method of the present invention, the porous layer has a sponge-like porous structure having a large number of pores as described above. Usually, this porous layer is freeze-dried or the like. If water removal is not performed by this, water is contained and the structure is hard in a water-containing state. In addition, a dried silk fibroin porous material can be obtained by freeze-drying the porous material.
本発明の製造方法により得られるシルクフィブロイン多孔質体は、引張弾性率で０．５〜７０．１ＭＰａという極めて高い強度を有するものであり、特許文献４において使用されている水溶性有機溶媒であるメタノール、エタノール、イソプロパノール、ブタノール、グリセロール、ジメチルスルフォキシド（ＤＭＳＯ）、ジメチルホルムアミド（ＤＭＦ）、ピリジン、アセトン、アセトニトリルを用いた多孔質体よりも優れた力学的特性を有している。 The silk fibroin porous material obtained by the production method of the present invention has a very high strength of 0.5 to 70.1 MPa in terms of tensile elastic modulus, and is a water-soluble organic solvent used in Patent Document 4. It has mechanical properties superior to porous materials using methanol, ethanol, isopropanol, butanol, glycerol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), pyridine, acetone, and acetonitrile.
多孔質体における多孔質層中の細孔直径は、上記したように好ましくは１〜３００μｍであり、シルクフィブロイン水溶液に加える脂肪族カルボン酸の種類、シルクフィブロイン水溶液と添加剤とを混合して得た溶液の濃度や該溶液を静置する時間や温度、静置後凝固温度まで冷却する際の降温速度などを変化させることである程度制御することが可能である。また、脂肪族カルボン酸を用いた場合には、１〜５０μｍという極めて小さいものも製造可能である。 As described above, the pore diameter in the porous layer in the porous body is preferably 1 to 300 μm, and is obtained by mixing the kind of aliphatic carboxylic acid added to the silk fibroin aqueous solution, the silk fibroin aqueous solution and the additive. It is possible to control to some extent by changing the concentration of the solution, the time and temperature at which the solution is allowed to stand, the cooling rate when cooling to the solidification temperature after standing. Moreover, when aliphatic carboxylic acid is used, a very small thing of 1-50 micrometers can be manufactured.
また、多孔質体の空孔率は、適宜調整可能であるが、通常５５％以上程度であり、好ましくは８０％以上と調整することもでき、より好ましくは９０％以上と調整することもできる。ここで、空孔率は、以下のようにして得られる値である。まず、得られた多孔質体を純水中に１日静置し完全に吸水させ、秤量した後（湿重量）、凍結乾燥して多孔質体中の水分を完全に除去し、再度秤量する（乾燥重量）。次いで、水の密度を１ｇ／ｃｍ3、フィブロインの密度を１．２ｇ／ｃｍ3、含水状態のフィブロイン多孔質体の密度を１ｇ／ｃｍ3と仮定し、次式に従って得られる値をシルクフィブロイン多孔質体の空孔率とした。
空孔率＝（湿重量−乾燥重量／１．２）／湿重量×１００
このように、本発明の製造方法で得られたシルクフィブロイン多孔質体は極めて大きい空孔率を有するものであり、様々な用途において、優れた性能を示すものである。
The porosity of the porous body can be adjusted as appropriate, but is usually about 55% or more, preferably 80% or more, more preferably 90% or more. . Here, the porosity is a value obtained as follows. First, the obtained porous body is left in pure water for 1 day to completely absorb water, weighed (wet weight), freeze-dried to completely remove water in the porous body, and weighed again. (Dry weight). Next, assuming that the density of water is 1 g / cm 3 , the density of fibroin is 1.2 g / cm 3 , and the density of the fibroin porous material in a water-containing state is 1 g / cm 3 , the value obtained according to the following equation is the silk fibroin porosity. The porosity of the material was taken.
Porosity = (wet weight−dry weight / 1.2) / wet weight × 100
Thus, the silk fibroin porous material obtained by the production method of the present invention has an extremely large porosity, and exhibits excellent performance in various applications.
以下に、本発明を実施例によりさらに具体的に説明するが、本発明は、これらの実施例によってなんら限定されるものではない。 EXAMPLES The present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
実施例１
（シルクフィブロイン溶液の調製）
シルクフィブロイン水溶液は、シルクフィブロイン粉末（ＫＢセーレン社製、商品名：「フィブロインＩＭ」）を９Ｍ臭化リチウム水溶液に溶解し、遠心分離で不溶物を除去したのち、超純水に対して透析を繰り返すことによって得た。得られたシルクフィブロイン水溶液を透析チューブ中で風乾し濃縮した。この濃縮液に添加剤として酢酸水溶液を添加し、シルクフィブロイン濃度が１０質量％、酢酸濃度が２質量％であるシルクフィブロイン溶液を調製した。
（多孔質体の製造）
このシルクフィブロイン溶液をアルミ板で作製した型（内側サイズ；８０ｍｍ×４０ｍｍ×４ｍｍ）に流し込み、低温恒温槽（ＥＹＥＬＡ社製ＮＣＢ−３３００）に入れて−５℃で２時間静置した。その後下記条件で凍結した。
（凍結条件）
凍結は、予め低温恒温槽の初期温度を−５℃に冷却しておいた低温恒温槽中にシルクフィブロイン溶液を入れた型を投入し、３℃／時間の速度で−２０℃まで冷却し、そのままの温度で５時間保持した。
凍結した試料を自然解凍で室温に戻してから、型から取り出し、超純水に浸漬し、超純水を１日２回、３日間交換することによって、使用した酢酸を除去した。
Example 1
(Preparation of silk fibroin solution)
Silk fibroin aqueous solution is prepared by dissolving silk fibroin powder (KB Selen, trade name: “Fibroin IM”) in 9M lithium bromide aqueous solution, removing insoluble matter by centrifugation, and then dialysis against ultrapure water. Obtained by repeating. The obtained silk fibroin aqueous solution was air-dried in a dialysis tube and concentrated. An acetic acid aqueous solution was added to the concentrate as an additive to prepare a silk fibroin solution having a silk fibroin concentration of 10% by mass and an acetic acid concentration of 2% by mass.
(Manufacture of porous material)
This silk fibroin solution was poured into a mold (inside size; 80 mm × 40 mm × 4 mm) made of an aluminum plate, placed in a low temperature thermostatic bath (NCB-3300 manufactured by EYELA), and allowed to stand at −5 ° C. for 2 hours. Thereafter, it was frozen under the following conditions.
(Freezing condition)
For freezing, a mold in which the silk fibroin solution is placed in a low-temperature thermostatic chamber that has been cooled to -5 ° C in advance is cooled to -20 ° C at a rate of 3 ° C / hour, The temperature was kept at that temperature for 5 hours.
The frozen sample was returned to room temperature by natural thawing, then removed from the mold, immersed in ultrapure water, and the acetic acid used was removed by exchanging the ultrapure water twice a day for 3 days.
得られたシルクフィブロイン多孔質体の構造を、走査型電子顕微鏡を用いて観察した。走査型電子顕微鏡は、Ｐｈｉｌｉｐｓ社製ＸＬ３０−ＦＥＧを使用して、低真空無蒸着モード、加速電圧１０ｋＶで測定を行った。なお、シルクフィブロイン多孔質体の構造は、多孔質体の表面（フィルム層）及び多孔質体を切断して露出させた内部（多孔質層）を観察した。フィルム層及び多孔質層の走査型電子顕微鏡写真を各々図２及び図３に示す。 The structure of the obtained silk fibroin porous material was observed using a scanning electron microscope. The scanning electron microscope was measured using a Philips XL30-FEG in a low vacuum non-deposition mode and an acceleration voltage of 10 kV. In addition, the structure of the silk fibroin porous body observed the surface (film layer) of the porous body, and the inside (porous layer) exposed by cutting the porous body. Scanning electron micrographs of the film layer and porous layer are shown in FIGS. 2 and 3, respectively.
（引張弾性率）
得られたシルクフィブロイン多孔質体の力学的特性を、ＩＮＳＴＲＯＮ社マイクロテスター５５４８型を用いて評価した。作製したシルクフィブロイン多孔質体から４０ｍｍ×４ｍｍ×４ｍｍの試験片を切り出し、この試験片を２ｍｍ／ｍｉｎの条件で引っ張った際の強度とひずみをグラフ化し、傾きから引張弾性率を求めた。その結果を表１に示す。なお、引張弾性率は、作製した多孔質体から５点の試験片を作製し、さらに異なる日に作製した多孔質体から５点の試験片を切り出し、それら１０点について測定を行った際の測定結果の平均値を示している。
(Tensile modulus)
The mechanical properties of the obtained silk fibroin porous material were evaluated using INSTRON Micro Tester Model 5548. A 40 mm × 4 mm × 4 mm test piece was cut out from the produced silk fibroin porous material, and the strength and strain when this test piece was pulled under the condition of 2 mm / min was graphed, and the tensile modulus was obtained from the slope. The results are shown in Table 1. The tensile elastic modulus was obtained when five test pieces were prepared from the prepared porous body, and further five test pieces were cut out from the porous bodies prepared on different days, and measurement was performed on these ten points. The average value of the measurement results is shown.
（空孔率）
得られた多孔質体を純水中に１日静置し完全に吸水させ、秤量した後（湿重量）、凍結乾燥して多孔質体中の水分を完全に除去し、再度秤量した（乾燥重量）。水の密度を１ｇ／ｃｍ3、フィブロインの密度を１．２ｇ／ｃｍ3、含水状態のフィブロイン多孔質体の密度を１ｇ／ｃｍ3と仮定し、次式に従ってシルクフィブロイン多孔質体の空孔率の測定を行った。
空孔率＝（湿重量−乾燥重量／１．２）／湿重量×１００
(Porosity)
The obtained porous body was left in pure water for 1 day to completely absorb water, weighed (wet weight), freeze-dried to completely remove water in the porous body, and weighed again (dried) weight). Assuming that the density of water is 1 g / cm 3 , the density of fibroin is 1.2 g / cm 3 , and the density of the fibroin porous material in a water-containing state is 1 g / cm 3 , the porosity of the silk fibroin porous material is calculated according to the following formula: Was measured.
Porosity = (wet weight−dry weight / 1.2) / wet weight × 100
実施例２〜３１及び比較例１〜１５
実施例１において、第１表に示されるシルクフィブロイン濃度、添加剤の種類、添加剤の濃度及び低温恒温槽の初期温度とした以外は実施例１と同様にしてシルクフィブロイン多孔質体を得た。得られた多孔質体について、実施例１と同様にして引張弾性率及び空孔率を測定した。これらの数値を第１−１〜１−３表に示す。また、実施例２〜６で得られた多孔質体の断面（多孔質層）を、走査型電子顕微鏡を用いて撮影した。撮影した走査型電子顕微鏡写真を各々図４〜８に示す。
Examples 2-31 and Comparative Examples 1-15
In Example 1, a silk fibroin porous material was obtained in the same manner as in Example 1 except that the silk fibroin concentration, the type of additive, the concentration of the additive, and the initial temperature of the low-temperature thermostatic bath shown in Table 1 were used. . About the obtained porous body, it carried out similarly to Example 1, and measured the tensile elasticity modulus and the porosity. These numerical values are shown in Tables 1-1 to 1-3. Moreover, the cross section (porous layer) of the porous body obtained in Examples 2-6 was image | photographed using the scanning electron microscope. The photographed scanning electron micrographs are shown in FIGS.
本発明の製造方法により得られるシルクフィブロイン多孔質体は、吸水性が高く、かつ安全性にも問題がないことから、保湿などを目的とした化粧品・エステ分野などに広く適用することができる。具体的には、ピーリングパックや化粧用パフとして好適に使用することができる。また、凍結に用いる容器の形状を変えることで、所望の形状のものを容易に得ることができることから、例えば、顔の形状に合わせたフェイスマスクとして好適に使用することができる。
また、本発明の製造方法により得られるシルクフィブロイン多孔質体は、吸水量を変えることでその重さを制御することができ、かつ安全性にも問題がないことから、例えば、内視鏡観察下で切除された生体組織を牽引するための重りとして、好適に使用し得る。
その他、癒着防止膜、創傷被覆材や薬剤徐放担体など、止血スポンジなどの医療分野、紙おむつや生理用品などの生活日用品分野、組織工学や再生医工学における細胞培養支持体や組織再生支持体、微生物や細菌などの住処になる支持体などに好適に使用することができる。
Since the silk fibroin porous material obtained by the production method of the present invention has high water absorption and no problem with safety, it can be widely applied in the cosmetics / esthetic field for the purpose of moisturizing. Specifically, it can be suitably used as a peeling pack or a cosmetic puff. Moreover, since the thing of a desired shape can be obtained easily by changing the shape of the container used for freezing, it can be used suitably as a face mask adapted to the shape of the face, for example.
Further, the silk fibroin porous material obtained by the production method of the present invention can control its weight by changing the amount of water absorption, and there is no problem in safety. It can be suitably used as a weight for pulling the living tissue excised below.
In addition, anti-adhesion membranes, wound dressings and sustained drug carriers, medical fields such as hemostatic sponges, daily necessities such as disposable diapers and sanitary products, cell culture supports and tissue regeneration supports in tissue engineering and regenerative medical engineering, It can be suitably used for a support that serves as a residence for microorganisms and bacteria.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010227030A JP5704688B2 (en) | 2010-10-06 | 2010-10-06 | Method for producing silk fibroin porous material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010227030A JP5704688B2 (en) | 2010-10-06 | 2010-10-06 | Method for producing silk fibroin porous material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2012082244A true JP2012082244A (en) | 2012-04-26 |
| JP5704688B2 JP5704688B2 (en) | 2015-04-22 |
Family
ID=46241477
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2010227030A Expired - Fee Related JP5704688B2 (en) | 2010-10-06 | 2010-10-06 | Method for producing silk fibroin porous material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5704688B2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012082246A (en) * | 2010-10-06 | 2012-04-26 | Hitachi Chemical Co Ltd | Silk fibroin porous body, and method for manufacturing the same |
| WO2015163293A1 (en) * | 2014-04-21 | 2015-10-29 | 日立化成株式会社 | Fibroin porous body, fibroin aqueous solution, and fibroin starting material |
| EP2990413A4 (en) * | 2013-04-25 | 2016-10-05 | Spiber Inc | Polypeptide porous body and method for producing same |
| US9732125B2 (en) | 2013-04-25 | 2017-08-15 | Spiber Inc. | Polypeptide particle and method for producing same |
| US9968682B2 (en) | 2013-04-25 | 2018-05-15 | Spiber Inc. | Polypeptide hydrogel and method for producing same |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01118544A (en) * | 1987-11-02 | 1989-05-11 | Agency Of Ind Science & Technol | Porous product of silk fibroin |
| JP2004018757A (en) * | 2002-06-19 | 2004-01-22 | National Institute Of Agrobiological Sciences | Biodegradable biopolymer material, method for producing the same and functional material composed of the polymer material |
| WO2009133532A1 (en) * | 2008-04-30 | 2009-11-05 | Oxford Biomaterials Limited | An implantable material and a method for the preparation thereof |
| JP2011231096A (en) * | 2010-04-06 | 2011-11-17 | Hitachi Chem Co Ltd | Sheet for skin care |
-
2010
- 2010-10-06 JP JP2010227030A patent/JP5704688B2/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01118544A (en) * | 1987-11-02 | 1989-05-11 | Agency Of Ind Science & Technol | Porous product of silk fibroin |
| JP2004018757A (en) * | 2002-06-19 | 2004-01-22 | National Institute Of Agrobiological Sciences | Biodegradable biopolymer material, method for producing the same and functional material composed of the polymer material |
| WO2009133532A1 (en) * | 2008-04-30 | 2009-11-05 | Oxford Biomaterials Limited | An implantable material and a method for the preparation thereof |
| JP2011231096A (en) * | 2010-04-06 | 2011-11-17 | Hitachi Chem Co Ltd | Sheet for skin care |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012082246A (en) * | 2010-10-06 | 2012-04-26 | Hitachi Chemical Co Ltd | Silk fibroin porous body, and method for manufacturing the same |
| EP2990413A4 (en) * | 2013-04-25 | 2016-10-05 | Spiber Inc | Polypeptide porous body and method for producing same |
| US9732125B2 (en) | 2013-04-25 | 2017-08-15 | Spiber Inc. | Polypeptide particle and method for producing same |
| US9968682B2 (en) | 2013-04-25 | 2018-05-15 | Spiber Inc. | Polypeptide hydrogel and method for producing same |
| US10065997B2 (en) | 2013-04-25 | 2018-09-04 | Spiber Inc. | Polypeptide porous body and method for producing same |
| WO2015163293A1 (en) * | 2014-04-21 | 2015-10-29 | 日立化成株式会社 | Fibroin porous body, fibroin aqueous solution, and fibroin starting material |
| JPWO2015163293A1 (en) * | 2014-04-21 | 2017-04-13 | 日立化成株式会社 | Fibroin porous material, fibroin aqueous solution and raw material for fibroin |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5704688B2 (en) | 2015-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5658659B2 (en) | Method for producing silk fibroin porous material | |
| JP5704688B2 (en) | Method for producing silk fibroin porous material | |
| Ceylan et al. | Effect of crosslinking methods on the structure and biocompatibility of polyvinyl alcohol/gelatin cryogels | |
| JP5700039B2 (en) | Silk fibroin porous material and method for producing the same | |
| RU2640024C2 (en) | Simple ethers of cellulose that have increased thermal strength of gel | |
| JP2012082245A (en) | Method for manufacturing silk fibroin porous body | |
| JP5565629B2 (en) | Method for producing silk fibroin porous material | |
| JP6205723B2 (en) | Porous material and porous sheet | |
| JP5704687B2 (en) | Silk fibroin porous material | |
| Cai et al. | Hierarchically porous films architectured by self-assembly of prolamins at the air–liquid interface | |
| JP2012082243A (en) | Method for manufacturing silk fibroin porous body | |
| JP5621487B2 (en) | Silk fibroin porous material and method for producing the same | |
| JP2012081593A (en) | Silk fibroin porous body | |
| JP2017048303A (en) | Silk fibroin powder | |
| WO2015162676A1 (en) | Porous material and porous sheet | |
| JP2011173963A (en) | Fibroin sponge sheet and method for producing the same | |
| JP2012082240A (en) | Silk fibroin porous body | |
| JP2012081594A (en) | Silk fibroin porous body | |
| JP5703211B2 (en) | Sponge manufacturing method | |
| JP6651757B2 (en) | Composite and method for producing the same | |
| JP6405895B2 (en) | Fibroin complex | |
| Alfata et al. | The Effect of Additional Zinc Oxide to Antibacterial Property of Chitosan/Collagen-Based Scaffold | |
| Naseri | Porous structures based on nanopolysaccharides for medical applications | |
| Imani et al. | Synthesis and Characterization of Biodegradable HemostatGelatin Sponge forSurgery Application | |
| JP6613548B2 (en) | Skin care material and skin care method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130906 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20130906 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20140417 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140507 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140707 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20150127 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20150220 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5704688 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313111 |
|
| R360 | Written notification for declining of transfer of rights |
Free format text: JAPANESE INTERMEDIATE CODE: R360 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313117 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313115 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: R3D02 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: R3D02 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313115 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |