Analysis of sulfated glycosaminoglycans in ECM scaffolds for tissue engineering applications: Modified alcian blue method development and validation

Tuyajargal Iimaa, Yasuhiro Ikegami, Ronald Bual, Nana Shirakigawa, Hiroyuki Ijima

研究成果: ジャーナルへの寄稿記事

1 引用 (Scopus)

抄録

Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 °C for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 v/v). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14-4.83% and 3.16-7.02% (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5%, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications.

元の言語英語
記事番号19
ジャーナルJournal of Functional Biomaterials
10
発行部数2
DOI
出版物ステータス出版済み - 1 1 2019

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A73025
Alcian Blue
Scaffolds (biology)
Tissue engineering
Heparin
Proteins
Scleroproteins
Ethanolamine
Density (optical)
Glycosaminoglycans
Complex Mixtures
Liver
Spectrometry
Coloring Agents

All Science Journal Classification (ASJC) codes

  • Biomaterials
  • Biomedical Engineering

これを引用

Analysis of sulfated glycosaminoglycans in ECM scaffolds for tissue engineering applications : Modified alcian blue method development and validation. / Iimaa, Tuyajargal; Ikegami, Yasuhiro; Bual, Ronald; Shirakigawa, Nana; Ijima, Hiroyuki.

:: Journal of Functional Biomaterials, 巻 10, 番号 2, 19, 01.01.2019.

研究成果: ジャーナルへの寄稿記事

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abstract = "Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 °C for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 v/v). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14-4.83{\%} and 3.16-7.02{\%} (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5{\%}, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications.",
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