Construction of Tough, in Situ Forming Double-Network Hydrogels with Good Biocompatibility
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Hydrogels are required to have high mechanical properties, biocompatibility, and an easy fabrication process for biomedical applications. Double-network hydrogels, although strong, are limited because of the complicated preparation steps and toxic materials involved. In this study, we report a simple method to prepare tough, in situ forming polyethylene glycol (PEG)-agarose double-network (PEG-agarose DN) hydrogels with good biocompatibility. The hydrogels display excellent mechanical strength. Because of the easily in situ forming method, the resulting hydrogels can be molded into any form as needed. In vitro and in vivo experiments illustrate that the hydrogels exhibit satisfactory biocompatibility, and cells can attach and spread on the hydrogels. Furthermore, the residual amino groups in the network can also be functionalized for various biomedical applications in tissue engineering and cell research.Keywords:
Biocompatibility
Agarose
Hydrogels are required to have high mechanical properties, biocompatibility, and an easy fabrication process for biomedical applications. Double-network hydrogels, although strong, are limited because of the complicated preparation steps and toxic materials involved. In this study, we report a simple method to prepare tough, in situ forming polyethylene glycol (PEG)-agarose double-network (PEG-agarose DN) hydrogels with good biocompatibility. The hydrogels display excellent mechanical strength. Because of the easily in situ forming method, the resulting hydrogels can be molded into any form as needed. In vitro and in vivo experiments illustrate that the hydrogels exhibit satisfactory biocompatibility, and cells can attach and spread on the hydrogels. Furthermore, the residual amino groups in the network can also be functionalized for various biomedical applications in tissue engineering and cell research.
Biocompatibility
Agarose
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Low Toxic Emulsifiable Concentrates of Pyrethroids with Polyethylene Glycol and Polypropylene Glycol
ポリエチレングリコール (PEG) またはポリプロピレングリコール (PPG) を用いて, ピレスロイドの乳剤化を検討した. PEGおよびPPGの平均分子量 (Mw) がおのおの150および134以上のとき, フェンプロパスリンは10%まで, エスフェンバレレートは20%まで相溶性が良好であった. また, PEGおよびPPGのMwがおのおの150～600および134～300のとき, フェンプロパスリン5, 10%乳剤およびエスフェンバレレート5, 10, 20%乳剤は, いずれも乳化安定性が良好であった. PPG (Mw=300) を溶剤としたフェンプロパスリン5%乳剤の乳化分散性は, 5～20℃では100%であったが, 250Cおよび30℃では約80%とやや低下した. この乳剤に少量の乳化剤を添加すると, 高温での乳化分散性が向上した. PEGまたはPPGを溶剤としたフェンプロパスリンおよびエスフェンバレレート5%乳剤とPEGを溶剤としたフェンバレレート, パーメスリン, d-フェノスリンおよびサイパーメスリン10%乳剤は, いずれも対応する通常の乳剤に比べ急性経口毒性が明らかに軽減された. PEGまたはPPGを溶剤としたエスフェンバレレート5%乳剤は, ウサギの眼に対する刺激性も軽減された. さらに, PEGまたはPPGを溶剤とした乳剤は, 臭気や引火性の面でも通常の乳剤より優れていた. これらの乳剤は, 保存安定性, 低温安定性, 自己乳化性, 生物効力も良好であった.
Polypropylene glycol
PEG 400
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PEG (polyethylene glycol) /Dex (dextran) およびPEG/PK (リン酸カリウム) 系による水性二相抽出法をパパイヤラテックスからのパパインの分離・精製に用いた.分配係数はパパインと分配系との間に作用する静電的, 立体的, 疎水的などの各種の相互作用に依存するが, PEG/PK系では, PEGとパパインとの疎水的相互作用の寄与が大きく, 分配係数と選択性はPEG/Dex系より高い.また, パパイヤラテックス粉末を水性二相系に直接添加し, 浸出と分離を同時に行わせる方式はパパイヤラテックスの浸出液を分配させる方式よりも有効である.PEGのOHをパパインと特異的アフィニティを持つ各種のリガンドで置換し, それらの修飾PEGを水性二相抽出に用いた.その結果, 分配係数と選択性は大きく向上するが, その結果はPB (Procion-Blue) -PEGを用いたPEG/PK系で顕著である.
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Fengci mulberry seeds being used as material,the effects of the different contents of polyethylene glycol(PEG)(0,1,2.5,5,7.5 and 10 g/L-simulated drought stress on mulberry seed germination and physiology were studied.The results showed that the germination velocity,ratio and potential of the seeds that were treated with 1 % PEG were higher than the CK,but those of other treatments were obviously decreased as the increase of the content of PEG.The seeds treated with 10 %PEG did not germinate after 5 days.The proline content was decreased when they were treated with low content of PEG and increased when they were treated with high content of PEG,but the content of dissoluble protein and sugar was increased along with the increase of the content of PEG.
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Extrusion-based 3D bioprinting enables the production of customized hydrogel structures that can be employed in flow reactors when printing with enzyme-containing inks. The present study compares inks based on either low-melt agarose or agar at different concentrations (3-6%) and loaded with the thermostable enzyme esterase 2 from the thermophilic organism Alicyclobacillus acidocaldarius (AaEst2) with regard to their suitability for the fabrication of such enzymatically active hydrogels. A customized printer setup including a heatable nozzle and a cooled substrate was established to allow for clean and reproducible prints. The inks and printed hydrogel samples were characterized using rheological measurements and compression tests. All inks were found to be sufficiently printable to create lattices without overhangs, but printing quality was strongly enhanced at 4.5% polymer or more. The produced hydrogels were characterized regarding mechanical strength and diffusibility. For both properties, a strong correlation with polymer concentration was observed with highly concentrated hydrogels being more stable and less diffusible. Agar hydrogels were found to be more stable and show higher diffusion rates than comparable agarose hydrogels. Enzyme leaching was identified as a major drawback of agar hydrogels, while hardly any leaching from agarose hydrogels was detected. The poor ability of agar hydrogels to permanently immobilize enzymes indicates their limited suitability for their employment in perfused biocatalytic reactors. Batch-based activity assays showed that the enzymatic activity of agar hydrogels was roughly twice as high as the activity of agarose hydrogels which was mostly attributed to the increased amount of enzyme leaching. Agarose bioinks with at least 4.5% polymer were identified as the most suitable of the investigated inks for the printing of biocatalytic reactors with AaEst2. Drawbacks of these inks are limited mechanical and thermal stability, not allowing the operation of a reactor at the optimum temperature of AaEst2 which is above the melting point of the employed low-melt agarose.
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Synthesis of a variety of 1,5-benzothiazepines using polyethylene glycol PEG-400 as a medium and promoter.The synthesis is carried out using ultrasonic irradiation.The advantage of this protocol is that it eco-friendly, mild reaction conditions and the synthesis highlights the use of ultrasound irradiation.
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Fe3+とM2+(M=Ni,Cu,Co,Zn)を含む複シュウ酸塩を,金属硝酸塩水和物のポリエチレングリコール(以後PEGと略す)溶液を353 K,3h加熱処理することで調製した。シュウ酸塩は,PEG溶液中のPEG-陽イオン複合体が硝酸イオンによって酸化されることで生成した。得られたシュウ酸塩の構造と陽イオン組成は,出発物質であるPEG溶液の組成だけでなく,PEG分子量にも依存した。PEG分子量の増加により,得られたシュウ酸塩の結晶化度や粒子の大きさが増大した。これらの事実を理解するためにPEG溶液中のPEG-陽イオン複合体の生成過程について二つのモデルを検討した。それらは,Fe3+とM2+のPEGへの配位が独立に起こると仮定するものと協同的に起こると仮定するものである。これらのモデルを用いてPEG溶液の陽イオン組成と得られたシュウ酸塩の陽イオン組成の間の関係式を導いた。モデルから導かれた関係と実験結果を比較した結果,Fe3+がPEGに配位するとき,その近傍で常にM2+のPEGへの配位が起こることがわかった。
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Molecular mass
Gel permeation chromatography
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Five agarose types (D1LE, D2LE, LM, MS8 and D5) were evaluated in tissue engineering and compared for the first time using an array of analysis methods. Acellular and cellular constructs were generated from 0.3–3%, and their biomechanical properties, in vivo biocompatibility (as determined by LIVE/DEAD, WST-1 and DNA release, with n = 6 per sample) and in vivo biocompatibility (by hematological and biochemical analyses and histology, with n = 4 animals per agarose type) were analyzed. Results revealed that the biomechanical properties of each hydrogel were related to the agarose concentration (p < 0.001). Regarding the agarose type, the highest (p < 0.001) Young modulus, stress at fracture and break load were D1LE, D2LE and D5, whereas the strain at fracture was higher in D5 and MS8 at 3% (p < 0.05). All agaroses showed high biocompatibility on human skin cells, especially in indirect contact, with a correlation with agarose concentration (p = 0.0074 for LIVE/DEAD and p = 0.0014 for WST-1) and type, although cell function tended to decrease in direct contact with highly concentrated agaroses. All agaroses were safe in vivo, with no systemic effects as determined by hematological and biochemical analysis and histology of major organs. Locally, implants were partially encapsulated and a pro-regenerative response with abundant M2-type macrophages was found. In summary, we may state that all these agarose types can be safely used in tissue engineering and that the biomechanical properties and biocompatibility were strongly associated to the agarose concentration in the hydrogel and partially associated to the agarose type. These results open the door to the generation of specific agarose-based hydrogels for definite clinical applications such as the human skin, cornea or oral mucosa.
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Agarose
Histology
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We have used two general approaches to facilit:1te the growth of protein c1ystals for X-ray diffi•action experiments.The first approach is called SAF, for smallest, active fi:agment.SAF is simply a logical extension of the old observation that proteolytic fi•agments of proteins often both harbour complete biological activity and form useful c1ystals more reaclily than the full length protein.However, rather than depend on the fortuitous position of protease recognition sites, SAF couples partial proteolysis with deletion analysis in order to refine the bmmdaries of the domain of interest.In practice, an iterative cycle of partial proteolysis, deletion analysis, biochemical assays, protein over-expression and c1ystallization is used to identify a domain that fonm diffraction-quality crystals.SAF capitalizes on the ease and rapidity of subcloning, overexpressing and pmifying proteins as well as the technical ease and availability ofN-tenninal sequencing and mass spectrometry.We have used the SAF approach for three proteins, and we have generated fragments of each that were suitable for str11cture detemrination.The second approach.lipid-layer seeding, uses lipid monolayers to seed c1ystal growth.We observed several years ago that two-din1ensional protein cryst:1ls grown on lipid layers effectively nucleate the epita\.ialgrowth of three din1ensional protein c1yst:1ls.In this way, crystals suitable for X -ray crystallography can be grow11 more rapidly, and at subst:mtially lower protein concentrations and precipit:mts than for conventional c1ystal 1:Iials.The methodology has now been developed such that the procedure takes only a few seconds per c1ystal trial.We are now applying the method to an assortment of proteins in an effort to demonstr•ate the generality of the approach.
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