Country：Japan

Country：Japan

Country：Japan

DOI： 10.1080/09168451.2020.1743167

強力なmiRNA阻害剤はアルゴノート2(Ago2)上でmiRNAと強く結合する必要があると推測し、その様な阻害剤を細胞内で選択することを試みた。Δ型オリゴ配列とd-21ライブラリーから作成したLidNAライブラリーを標的のmiR-21とともにHEK293T細胞に導入して培養し、その細胞融解物から抗Ago2抗体でLidNA+Ago2複合体を分離・選択した。このサイクルを4回行い、選択したLidNAのmiR-21阻害活性を比較した。その結果、強力な阻害剤は細胞内でゆっくりと分解するが、弱い活性を持つ阻害剤は迅速に細胞内で分解することが判明した。miRNA結合領域周囲の選択された配列を組み合わせると、miRNA阻害活性を促進させることが出来ると考えられた。

DOI： 10.1080/09168451.2020.1734443

DOI： 10.1089/mab.2020.0007

miRNAの機能を阻害するDNA(LidNA)を作成するため、非修飾DNAを利用し、長い二本鎖領域の代わりに二本鎖領域を2つ持ったLidNAを作成し、検討した。その結果、LidNAは2'-O-メチル化RNAや架橋型人工核酸よりも強くmiRNA活性を阻害した。LidNAを改良するために、2つの二本鎖領域を結合して、Δ様の形になるような分子を作り、デルタ型LidNAと命名した。内因性と外因性のmiRNAを標的としたデルタ型LidNAを作成したところ、少なくとも10日間強力なmiRNA阻害効果を示した。miR-21を標的としたデルタ型LidNA-21は癌細胞の増殖を阻害した。この新しく開発したLidNAは多くの領域のmiRNA研究にも役立つと考えられた。

DOI： 10.1002/1873-3468.13756

スフェロイドを得るための、実験室で簡便に作れる培養皿の作成を試みた。キトサンを無水酢酸と反応させてキチンゲルを作成した。このゲルを乾燥させ、キチンシートを作成した。HEK293T細胞をキチンシートで培養したところ、スフェロイドが形成された。この細胞は丸い形をしており、キチンシートには付着していなかった。しかし、タイムラプスカメラで長時間撮影したところ、視野範囲に留まっており、キチンシートに弱く付着していると推定された。細胞が付着しない市販のPrimeSurfaceで培養したところ、スフェロイドは急速に移動し、スフェロイドのサイズもばらばらで、細胞の形もゆがんでいた。キチンシートを用いて、ゼラチン培地で培養またはキチンシートに付着しないL929細胞と共培養したところ、均一なスフェロイドが形成された。

DOI： 10.1016/j.bbrc.2020.02.010

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

フルオレセインイソチオシナート(FITC)で処理し、青色光照射し、架橋アルブミン層を親水性にして細胞パターニングする方法を開発した。架橋アルブミンコート皿を種々の濃度のFITCで処理し、青色光照射してL929細胞を培養したところ、高濃度のFITC処理域で細胞接着が観察された。低濃度(0.2mg/mL)のFITCで処理し青色光照射したところ、L929細胞は接着しなかったが、HepG2細胞は接着した。このシステムにより、細胞パターニングを解析することができると考えられた。

幼若期にX線被ばくを受けたマウス脾臓細胞への放射線誘発変異に対し、成熟期でのカロリー制限(CR)でも効果が得られるか実験した。雄性B6C3F1 gpt deltaマウスを用いて、X線被ばく群には1週齢期に3.8GyのX線照射を与え、4週間で離乳後、7週目までは標準食が与えられた。7週目からは無作為に(i)非照射で非CR餌(95kcal/週)が与えられた群、(ii)非照射でCR餌(65kcal/週)が与えられた群、(iii)3.8Gy照射を受け非CR餌(95kcal/週)が与えられた群、(iv)3.8Gy照射を受けCR餌(95kcal/週)が与えられた群の4群に分け実施した。CR開始前の7週齢、8週齢、100日齢に屠殺したマウスから脾臓、肝臓、肺、胸腺を摘出し、凍結保存後、gptアッセイにより点突然変異を検出した。その結果、脾臓由来DNAからgpt遺伝子の点突然変異が検出され、CR群の変異頻度は非CR群に比べ低下が認められ、配列解析からG:C→T:Aへの変異が抑制されていることが明らかにされた。以上の実験結果から、成熟期から開始したCRでも、酸化ストレスを低減されることにより、X線被ばくマウスの遺伝子変異頻度の低減が得られることが立証された。

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.jbiosc.2019.07.002

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Crosslinked albumin films, to which L929 cells do not attach by nature, acquire the L929-cell-adhesion capability by irradiation of an atmospheric-pressure plasma jet (APPJ) of He gas. The number of attached cells was 2.6 × 10⁴cells/cm²after the APPJ irradiation for 180 s, while conventional UV photolithography, which was performed in our previous work, required 2 h to obtain the same order of magnitude of the number of attached cells. The contact angle of samples decreased steeply from 105 to 38° in the first 10 s irradiation, but decreased quite gradually from 38 to 32° with increasing irradiation time from 10 to 180 s. In contrast to the nonlinear variation in the contact angles, the number of attached cells almost linearly increased from 4.5 × 10³to 2.6 × 10⁴cells/cm²with increasing treatment time. X-ray photoelectron spectroscopy of the samples indicated that hydrophilic functional groups of C–C=O gradually formed with increasing APPJ irradiation time up to 180 s. These results suggest that the cell-adhesion capability of the crosslinked albumin films is not simply explained by the decrease in contact angle but also by the formation of oxidized functional groups on the surface. The effects of UV and vacuum UV light from APPJ were negligible, which indicates that the formation of oxidized functional groups on the surface is not caused by photon-assisted surface reactions but by reactions involving chemically active species supplied from APPJ.

DOI： 10.7567/JJAP.55.07LG03

CiNii Article

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1021/acs.langmuir.5b03958

Publishing type：Research paper (scientific journal)  

We discovered the unique cell adhesive properties of ultraviolet (UV)-irradiated albumin films. Albumin films prepared using a cross-linking reagent with epoxy groups maintained native albumin properties, such as resistance to cell adhesion. Interestingly, the cell adhesive properties of films varied depending upon the UV irradiation time
specifically, cell adhesiveness increased until 2 h of UV irradiation, when the cell number attached to the film was similar to that of culture dishes, and then cell adhesiveness decreased until 20 h of UV irradiation, after which the surface returned to the initial non-adhesive state. To elucidate the molecular mechanisms underlying this phenomenon, we examined the effect of UV irradiation on albumin film properties. The following changes occurred in response to UV irradiation: decreased α-helical structure, cleavage of albumin peptide bonds, and increased hydrophilicity and oxygen content of the albumin film surface. In addition, we found a positive correlation between the degree of cell adhesion and the amount of fibronectin adsorbed on the film. Taken together, UV-induced changes in films highly affect the amount of cell adhesion proteins adsorbed on the films depending upon the irradiation time, which determines cell adhesion behavior.

DOI： 10.1021/acs.langmuir.5b03958

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Keratin was extracted as a reduced form from wool, which was then subjected to acetamidation, carboxymethylation or aminoethylation at abundant free cysteine residues to give acetamidated keratin (AAK), carboxymethylated keratin (CMK) and aminoethylated keratin (AEK). Hydrogels were prepared from intact and three chemically modified keratins simply by concentrating their aqueous solution and subsequent cooling. The lowest concentration to form a hydrogel without fluidity was 110 mg/ml for AAK, 120 mg/ml for AEK, 130 mg/ml for keratin and 180 mg/ml for CMK. Comparing with a hydrogel just prepared (swelling ratio: 600-700), each hydrogel slightly shrank in an acidic solution. While AAK hydrogel little swelled in neutral and basic solutions, other hydrogels became swollen and CMK hydrogel reached to dissolution. Hydrogels of keratin, AAK and AEK were found to support cell proliferation, although cell elongation on AAK and AEK hydrogel was a little suppressed. On the other hand, CMK hydrogel did not seem to be suitable for a cell substrate because of its high swelling in culture medium. Evaluation of the hydrogels as a drug carrier showed that keratin and AAK hydrogels were good sustained drug release carriers, which showed the drug release for more than three days, while the release from AEK and CMK hydrogels completed within one day. Thus, keratin and chemically modified keratin hydrogels, especially keratin and AAK hydrogels, were promising biomaterials as a cell substrate and a sustained drug release carrier.

CiNii Article

Publishing type：Research paper (scientific journal)  

Keratin was extracted as a reduced form from wool, which was then subjected to acetamidation, carboxymethylation or aminoethylation at abundant free cysteine residues to give acetamidated keratin (AAK), carboxymethylated keratin (CMK) and aminoethylated keratin (AEK). Hydrogels were prepared from intact and three chemically modified keratins simply by concentrating their aqueous solution and subsequent cooling. The lowest concentration to form a hydrogel without fluidity was 110 mg/ml for AAK, 120 mg/ml for AEK, 130 mg/ml for keratin and 180 mg/ml for CMK. Comparing with a hydrogel just prepared (swelling ratio: 600-700), each hydrogel slightly shrank in an acidic solution. While AAK hydrogel little swelled in neutral and basic solutions, other hydrogels became swollen and CMK hydrogel reached to dissolution. Hydrogels of keratin, AAK and AEK were found to support cell proliferation, although cell elongation on AAK and AEK hydrogel was a little suppressed. On the other hand, CMK hydrogel did not seem to be suitable for a cell substrate because of its high swelling in culture medium. Evaluation of the hydrogels as a drug carrier showed that keratin and AAK hydrogels were good sustained drug release carriers, which showed the drug release for more than three days, while the release from AEK and CMK hydrogels completed within one day. Thus, keratin and chemically modified keratin hydrogels, especially keratin and AAK hydrogels, were promising biomaterials as a cell substrate and a sustained drug release carrier. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2014.12.005

羊毛から還元してケラチン(K)を抽出し、アセトアミド化ケラチン(AAK)、カルボキシメチル化ケラチン(CMK)、アミノエチル化ケラチン(AEK)を調製した。その水溶液を濃縮・冷却してヒドロゲルを作成した。調製直後のヒドロゲルと比較して、酸性溶液ではそれぞれのヒドロゲルはわずかに収縮した。一方、中性とアルカリ性溶液では、AAKヒドロゲルはわずかしか膨潤しなかったが、他のヒドロゲルは膨潤し、CMKヒドロゲルは溶解した。K、AAKとAEKのヒドロゲルは細胞増殖をサポートしたが、AAKとAEKのヒドロゲル上の細胞の伸長はわずかに抑制された。CMKヒドロゲルは細胞基質としては不適切であった。薬物の担体として評価したところ、KとAAKのヒドロゲルは3日間薬物を放出し続け、良好な薬物徐放担体であったが、AEKとCMKのヒドロゲルは1日以内で放出してしまうため、不適切であった。

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

LidNA, a microRNA inhibitor consisting of a microRNA binding ssDNA region sandwiched between dsDNA regions had higher affinity to target oligonucleotides than that without dsDNA region. This enhancement in affinity was found to be owing to the suppressed mobility of ssDNA region by the presence of dsDNA regions.

CiNii Article

腫瘍では多くのmicroRNA(miRNA)が過剰発現しており、miRNA阻害剤であるmiRNAと相補的なアンチセンスオリゴヌクレオチドは、腫瘍の候補治療薬として期待される。以前、新規miRNA阻害剤として、miRNAに相補的な配列を持ち、その両端にdsDNAを付加した未修飾のDNAからなるLidNAを開発した。表面プラズモン共鳴アッセイを用いてmiRNAと相補的なオリゴヌクレオチドの結合能に対し、dsDNAが与える影響を調べたところ、dsDNA領域の付加によ16DNAに対する未修飾のオリゴヌクレオチドの結合能は著明に向上した。今回、そのメカニズムについて、miRNAの末端塩基と隣接する2本鎖領域の末端塩基間のスタッキングと、2本鎖領域の付加によるmiRNA結合領域の構造の安定化の2つの可能性を基に、表面プラズモン共鳴アッセイと蛍光偏光法にて検討した。その結果、結合親和性の亢進は、塩基スタッキングよりもmiRNA結合領域の移動性を抑制したことによるものであった。

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.msec.2014.04.016

Publishing type：Research paper (scientific journal)  

Infection after artificial joint replacement is a serious problem, which requires the re-implantation of prosthesis. To aim at developing bone filling materials having both osteoconductivity and ability as a sustained drug release carrier, composites of wool keratin or carboxymethylated (CM) keratin hydrogels with hydroxyapatite were prepared and evaluated as a sustained drug release carrier. CM-keratin was prepared by the reaction of keratin extracted from wool with iodoacetic acid. Hydrogels were obtained by dropping keratin or CM-keratin solution into CaCl2 solution. The composites were obtained by immersing hydrogels in simulated body fluid (SBF). The introduction of carboxymethyl groups to keratin facilitated the deposition of hydroxyapatite on hydrogel. After 7 days of immersion in SBF, a 4-5 times higher amount of hydroxyapatite was accumulated on CM-keratin hydrogel than that on keratin hydrogel. When salicylic acid was loaded on keratin and CM-keratin hydrogels, a good sustained release was observed
that is, 90% of a drug was released up to 14 days after 60 and 45% of the initial burst in 1 day. On the other hand, initial release within 1 day was suppressed by forming a composite with hydroxyapatite and the release was almost ceased at 3 days when 60% of the drug was released. Although further improvement to prolong drug release might be necessary, CaKHA and CaCMKHA are expected to be a promising novel type of bone filling materials which has both osteoconductivity and sustained drug release ability. © 2014 Elsevier B.V.

DOI： 10.1016/j.msec.2014.04.016

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Small interfering RNAs (siRNAs) are potent tools in biomedical research, which can reduce the expression level of target proteins through RNAi pathway. They are composed of 19-25 by double strand RNA (dsRNAs), therefore, stimulate dsRNAs dependent interferon responses in a non-specific manner. This problem has prevented siRNAs from being applied as new therapeautic agents. In the present paper, we tried to circumvent interferon responses using RNA/DNA hetero siRNAs (HsiRNAs) composed of RNA guide and DNA passenger strands. It was previously reported that siRNAs which were partially substituted with DNA had RNAi activity and that DNA substitution often caused the activity loss. In our results, HsiRNAs, in which the passenger strand of siRNAs were exchanged with DNA also showed much lower activity than that of parental siRNAs. Here, we found that attachment of 5' flanking sequence to DNA passenger strand improved the activity of HsiRNAs. Furthermore, the effective HsiRNAs induced much lower interferon responses than parental siRNAs. Thus, HsiRNAs with 5' flanking sequence are expected to be novel siRNA drug candidates.

CiNii Article

RNAガイド鎖とDNAパッセンジャー鎖から成るRNA/DNAヘテロsiRNA(HsiRNA)を用いてインターフェロン応答性を回避することを試みた。部分的にDNAに置換させたsiRNAは、DNA置換によってRNA干渉(RNAi)活性の消失が多く生じることが報告されていたが、siRNAのパッセンジャー鎖をDNAに置換したHsiRNAは、siRNAよりもさらに活性が低下したにもかかわらず、5'フランキング配列を付加してDNAパッセンジャー鎖の長さを延長するとHsiRNAの活性が高くなることが明らかになった。また、このHsiRNAは、インターフェロン応答性もsiRNAより低く抑えることができた。この5'フランキング配列を付加したHsiRNAは、新規siRNA医薬品の候補物質になりうると考えられた。

Publishing type：Research paper (scientific journal)  

Small interfering RNAs (siRNAs) are potent tools in biomedical research, which can reduce the expression level of target proteins through RNAi pathway. They are composed of 19-25 bp double strand RNA (dsRNAs), therefore, stimulate dsRNAs dependent interferon responses in a non-specific manner. This problem has prevented siRNAs from being applied as new therapeautic agents. In the present paper, we tried to circumvent interferon responses using RNA/DNA hetero siRNAs (HsiRNAs) composed of RNA guide and DNA passenger strands. It was previously reported that siRNAs which were partially substituted with DNA had RNAi activity and that DNA substitution often caused the activity loss. In our results, HsiRNAs, in which the passenger strand of siRNAs were exchanged with DNA also showed much lower activity than that of parental siRNAs. Here, we found that attachment of 5' flanking sequence to DNA passenger strand improved the activity of HsiRNAs. Furthermore, the effective HsiRNAs induced much lower interferon responses than parental siRNAs. Thus, HsiRNAs with 5' flanking sequence are expected to be novel siRNA drug candidates. © 2013 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2013.09.012

PubMed

Publishing type：Research paper (scientific journal)  

LidNA, a microRNA inhibitor consisting of a microRNA binding ssDNA region sandwiched between dsDNA regions had higher affinity to target oligonucleotides than that without dsDNA region. This enhancement in affinity was found to be owing to the suppressed mobility of ssDNA region by the presence of dsDNA regions. © 2014 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2014.02.023

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.febslet.2012.04.013

Publishing type：Research paper (scientific journal)  

Many miRNA inhibitors have been developed and they are chemically modified oligonucleotides such as 2′-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA was not yet reported as a miRNA inhibitor because of the low affinity of DNA/miRNA compared to mRNA/miRNA. We designed a structured unmodified DNA that significantly inhibits miRNA function. The clue structure for activity is the miRNA binding site between double stranded regions which is responsible for the miRNA inhibitory activity and tight binding to miRNA. We developed the miRNA inhibitor constructed with unmodified DNA, and named it LidNA, DNA that puts a lid on miRNA function. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2012.04.013

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

Publishing type：Research paper (international conference proceedings)  

Since 1985, HA granules were interposed on the interface between bone and bone cement at the cementation (Interface Bioactive Bone Cement : IBBC) in THA to prevent generation of connective tissue and osteolysis. To prevent infection, β-TCP impregnated with antibiotics along with HA granules was used. As TCP is resorbable, antibiotic release can be controlled. β-TCP granules were impregnated with antibiotics of folmoxef sodium (F), Vancomycine hydrochloride (V) cefortiam dihydrochloride (C) and cefozopran hydrochloride (CE). Three models of antibiotic release were assumed. Model [1] was antibiotic release from surroundings of β-TCP granules. Model [2] was the condition loaded under normal and reduced pressure. In Model [3], β-TCP was dissolved gradually in EDTA, as the model in the living body. In model [1], the amount of release of F, V and C was 3280, 300 and 3 μg, respectively and completed in 30 hours. In model [2], the amount of release of F, V, C and CE was 16, 8, 0 and 8000 μg in reduced pressure, respectively. The release of F, V and C completed within 24 hours and that of CE was in 6 days. In model [3], released amount of C and CE was 116 and 7100 μg, respectively and completed in 19 days. © (2012) Trans Tech Publications.

DOI： 10.4028/www.scientific.net/KEM.493-494.186

Publishing type：Research paper (international conference proceedings)  

Antibiotics release impregnated in HA granules, which were used in IBBC to prevent infection after total joint arthroplasty, was measured. For antibiotics, Flumarin, Vancomycin, Pansporin and Firstcin were used. Two models of antibiotics release were assumed
Model [I]: antibiotics release from surroundings of HA granules immediately after surgery and Model [II]: antibiotics release loaded on HA after antibiotics release from surroundings of HA granules as follows
(1) loading in normal pressure and (2) loading in reduced pressure. The amount of antibiotics loaded on HA is higher when loading is conducted under reduced pressure than that under normal pressure. Firstcin showed the highest loaded amount and the most desirable sustained release pattern. The antibiotics release from HA are varid depending on the antibiotics used. © (2012) Trans Tech Publications.

DOI： 10.4028/www.scientific.net/KEM.493-494.361

CiNii Article

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.msec.2010.02.020

Publishing type：Research paper (scientific journal)  

Serum albumin acts as a physiological carrier for various compounds including drugs. A hydrogel consisting of recombinant human serum albumin (rHSA) was prepared to take advantage of drug binding ability of albumin for a sustained drug release carrier. The hydrogel was prepared by mixing rHSA and dithiothreitol and casted to a polystyrene mold. Hydrogel formation was thought to occur through the intermolecular interaction of the hydrophobic groups by protein denaturation. The release of sodium benzoate and salicylic acid from the hydrogel completed in 2 h, while warfarin release continued for 24 h. The total amounts of the drugs released from 100 mg of 15 and 5% rHSA hydrogel were 2.3 and 1.4 mu mol for warfarin, 1.4 and 1.1 mu mol for salicylic acid and 0.9 and 0.9 mu mol for sodium benzoate. These results reflected the order of the binding ability of drugs for intact albumin indicating that the drug binding ability of HSA still remained after the hydrogel formation. However, fibroblast cells attached and proliferated well on the hydrogel, indicating that denaturation of rHSA proceeded to the extent to allow the cell attachment. The present rHSA hydrogel might be suitable for a sustained release carrier of drugs having affinity for albumin. (c) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.msec.2010.02.020

CiNii Article

CiNii Article

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.

CiNii Article

Publishing type：Research paper (scientific journal)  

Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.01.019

CiNii Article

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.msec.2007.11.008

Publishing type：Research paper (scientific journal)  

Novel fabrication method of highly porous and flexible keratin sponges was developed by combining a particulate-leaching method and a freeze-drying method. Reduced keratin aqueous solution was mixed with dried calcium alginate beads and was lyophilized to give keratin/calcium alginate complex, which was subsequently treated with EDTA solution to leach out calcium alginate beads. The resultant keratin sponge was flexible enough to handle even in dried state because of its quite high porosity (98.9 +/- 0.1%), which was brought about by the large and small pores formed by the elimination of calcium alginate beads and water. The sponge supported the attachment and the proliferation of mouse fibroblast cells. Thus, the keratin sponge given by the present fabrication method afforded one alternative as a cell scaffold for tissue engineering. (C) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.msec.2007.11.008

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

Collagen/calcium phosphate composite materials were prepared using an alkaline phosphatase-catalyzed hydrolysis of water-soluble phosphate esters in the presence of calcium ions. We prepared a collagen/calcium phosphate composite film by adding sequentially calcium chloride, disodium glycerophosphate and alkaline phosphatase to collagen solution and subsequent drying of the resultant composite suspension in a template. The calcium phosphate contained in the film was determined to be mainly hydroxyapatite from FT-IR and X-ray diffraction analysis. The film little swelled in water to keep its original morphology, while a collagen film became solubilized. This suggests the binding between collagen and calcium phosphate in complex materials. When the preosteoblast cell line, MC3T3-E1 cells were cultured on the film, the film was found to support the osteoblastic differentiation from the measurement of alkaline phosphatase activity., which is an early differentiation marker.

Collagen/calcium phosphate composite materials were prepared using an alkaline phosphatase-catalyzed hydrolysis of water-soluble phosphate esters in the presence of calcium ions. We prepared a collagen/calcium phosphate composite film by adding sequentially calcium chloride, disodium glycerophosphate and alkaline phosphatase to collagen solution and subsequent drying of the resultant composite suspension in a template. The calcium phosphate contained in the film was determined to be mainly hydroxyapatite from FT-IR and X-ray diffraction analysis. The film little swelled in water to keep its original morphology, while a collagen film became solubilized. This suggests the binding between collagen and calcium phosphate in complex materials. When the preosteoblast cell line, MC3T3-E1 cells were cultured on the film, the film was found to support the osteoblastic differentiation from the measurement of alkaline phosphatase activity, which is an early differentiation marker.<br>

DOI： 10.2109/jcersj2.116.10

CiNii Article

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.msec.2006.10.008

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.msec.2006.10.009

Publishing type：Research paper (scientific journal)  

To take advantage of the drug-binding ability of albumin as a component of drug delivery system, we have prepared hydrogels consisting of alginic acid (AL) and recombinant human serum albumin (rHSA) by dehydrating condensation using N-hydroxysuccininimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As rHSA content increased, the swelling ratio of the hydrogel decreased, indicating rHSA functioned as a crosslinker. In fact, trypsin treatment solubilized the hydrogel. Salicylic acid, which has high affinity for rHSA, was loaded most on the hydrogel of the highest rHSA content despite the lowest swelling ratio. Meanwhile, drugs with less affinity for HSA such as o-anisic acid and benzoic acid were preferably loaded on the hydrogel having the highest swelling ratio but the lowest HSA content. The release of salicylic acid from the hydrogel sustained longer than o-anisic acid and benzoic acid, reflecting the affinity of the drug for HSA. Furthermore, the hydrogel could carry much of positively charged dibucaine by the interaction with anionic alginic acid and showed highly sustained release. Since the safety of AL and rHSA in medical use is guaranteed, rHSA-crosslinked AL hydrogel is expected to use as a sustained drug release carrier for drugs having affinity for HSA and those with cationic charge. (C) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.msec.2006.10.008

Publishing type：Research paper (scientific journal)  

Poly(acrylamide) hydrogels crosslinked by bovine serum albumin (BSA) were prepared by the introduction of vinyl groups into BSA and subsequent co-polymerization with acrylamide (AAm). The bydrogels were loaded with four structurally resembled benzoic acid derivatives such as salicylic acid, o-anisic acid, salicylamide and sodium benzoate, and their release from the hydrogel was investigated. The affinity of these four compounds for BSA gradually decreased in a following order; salicylic acid &gt; o-anisic acid &gt; salicylamide &gt; sodium benzoate. The amounts of compounds loaded on a hydrogel of high BSA content and the duration of their release were found to be dependent on the affinity for BSA clearly reflecting the subtle difference in BSA affinity of each compound. Therefore, this hydrogel would be used not only as a sustained drug release carrier, but also a facile tool to evaluate the binding of various compounds to serum albumin. (C) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.msec.2006.10.009

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.ab.2006.10.004

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ab.2006.10.004

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

A keratin sponge was chemically modified to obtain carboxyl and amino sponges by the alkylation of a large amount of active SH group on keratin proteins with iodoacetic acid and 2-bromoethylamine, respectively. The carboxyl sponge having a large amount of carboxyl group was a scaffold that could bind significant amounts of basic bioactive proteins, such as lysozyme and bone morphogenetic protein (BMP)-2, and drugs. Lysozyme (maximum 3.7mg), a model of basic cytokines such as BMP-2, was absorbed by the carboxyl sponge (4.8mg), but not by the amino sponge. The lysozyme was rapidly released from the carboxyl sponge in a buffer containing at a concentration higher than 0.5M, but at 0.15M, near the physiological ionic strength, after initial burst (only 11%), no significant release was observed (15%, 48h). BMP-2 also bound the carboxyl sponge. The preosteoblast cells grown inside the BMP-2-loaded sponge differentiated, whereas those grown outside the sponge did not, suggesting that no significant amount of BMP-2 leaked into the surrounding media. The effects of BMP-2 were confined inside modified keratin sponge. Therefore, using in vivo, we expect that only internal osteogenesis will be induced and that no external heterotopis ossification will be induced.

CiNii Article

Publishing type：Research paper (scientific journal)  

A keratin sponge was chemically modified to obtain carboxyl and amino sponges by the alkylation of a large amount of active SH group on keratin proteins with iodoacetic acid and 2-bromo-ethylamine, respectively. The carboxyl sponge having a large amount of carboxyl group was a scaffold that could bind significant amounts of basic bioactive proteins, such as lysozyme and bone morphogenetic protein (BMP)-2, and drugs. Lysozyme (maximum 3.7 mg), a model of basic cytokines such as BMP-2, was absorbed by the carboxyl sponge (4.8 mg), but not by the amino sponge. The lysozyme was rapidly released from the carboxyl sponge in a buffer containing at a concentration higher than 0.5 M, but at 0.15 M, near the physiological ionic strength, after initial burst (only 11%), no significant release was observed (15%, 48 h). BMP-2 also bound the carboxyl sponge. The preosteoblast cells grown inside the BMP-2-loaded sponge differentiated, whereas those grown outside the sponge did not, suggesting that no significant amount of BMP-2 leaked into the surrounding media. The effects of BMP-2 were confined inside modified keratin sponge. Therefore, using in vivo, we expect that only internal osteogenesis will be induced and that no external heterotopis ossification will be induced.

DOI： 10.1263/jbb.102.425

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Albumin is the major plasma protein and acts as a physiological carrier for various compounds including drugs. To take advantage of the drug-binding ability of albumin for a drug delivery system, we have prepared hydrogels consisting of acrylamide (AAm) and bovine serum albumin (BSA) by introducing three to four vinyl groups into one BSA molecule and subsequently copolymerizing it with AAm. The resultant hydrogel was solubilized by trypsin treatment, since BSA served as a crosslinker in the hydrogel. The BSA-crosslinked hydrogel (BSA-AAm hydrogel) was loaded with salicylic acid or sodium benzoate and their release was investigated. The BSA-AAm hydrogel released much more salicylic acid than sodium benzoate. In addition, the amount of released salicylic acid increased with the BSA content of the hydrogel, despite a decrease in the swelling ratio of the hydrogel. On the other hand, the amount of released sodium benzoate increased with the swelling ratio. When a hydrogel crosslinked with N, N'-methylenebis (acrylamide) was used as a control, both drugs showed release tendencies similar to that of sodium benzoate from the BSA-AAm hydrogel. Furthermore, the salicylic acid release was sustained longer on the BSA-AAm hydrogel than the sodium benzoate release. Taken together, it is thought that albumin in the BSA-AAm hydrogel preferentially adsorbs salicylic acid and contributes to the high drug loading and the sustained release of salicylic acid.

CiNii Article

Publishing type：Research paper (scientific journal)  

Albumin is the major plasma protein and acts as a physiological carrier for various compounds including drugs. To take advantage of the drug-binding ability, of albumin for a drug delivery system, we have prepared hydrogels consisting of acrylamide (AAm) and bovine serum albumin (BSA) by introducing three to four vinyl groups into one BSA molecule and subsequently copolymerizing it with AAm. The resultant hydrogel was solubilized by trypsin treatment, since BSA served as a crosslinker in the hydrogel. The BSA-crosslinked hydrogel (BSA-AAm hydrogel) was loaded with salicylic acid or sodium benzoate and their release was investigated. The BSA-AAm hydrogel released much more salicylic acid than sodium benzoate. In addition, the amount of released salicylic acid increased with the BSA content of the hydrogel, despite a decrease in the swelling ratio of the hydrogel. On the other hand, the amount of released sodium benzoate increased with the swelling ratio. When a hydrogel crosslinked with N,N'-methylenebis (acrylamide) was used as a control, both drugs showed release tendencies similar to that of sodium benzoate from the BSA-AAm hydrogel. Furthermore, the salicylic acid release was sustained longer on the BSA-AAm hydrogel than the sodium benzoate release. Taken together, it is thought that albumin in the BSA-AAm hydrogel preferentially adsorbs salicylic acid and contributes to the high drug loading and the sustained release of salicylic acid.

DOI： 10.1263/hbb.100.551

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.biomaterials.2004.02.032

Publishing type：Research paper (scientific journal)  

Wool keratin sponges were reported to be useful scaffolds for long-term and high-density cell cultivation (J. Biotechnol. 93 (2002) 165). The hybrid of the keratin sponges with calcium phosphate materials gave the additional function. Two rapid fabrication methods for calcium phosphate hybrid biomaterials were described. Firstly, the CaP-precipitated sponges were obtained by only the immersion of the carboxyl-sponges, chemically introduced with high amount of carboxyl groups on the sponges, in calcium and phosphate ions containing buffers such as PBS(+) for only 1-3 days. Neither sponge, introduced with amino or amido groups or non-treated, gave significant calcium phosphate precipitation. The carboxyl-sponges were mimics of matrix gamma-carboxyglutamic acid protein, which is responsible for osteoblast calcification. Secondly, the hydroxyapatite particle suspension was added onto carboxyl-sponges to fabricate trapped sponge. The trapped hydroxyapatite particles might interact with keratin protein of the sponge walls. Preliminary experiments measuring the expression of alkaline phosphatase, early osteoblast differentiation marker, suggested that both hybrid sponges, CaP-precipitated and trapped sponges, alter the differentiation pattern of preosteoblasts, MC3T3-E1. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biomaterials.2004.02.032

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (other)   Kind of work：Single Work  

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