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Open AccessArticle

Analysis of Sulfated Glycosaminoglycans in ECM Scaffolds for Tissue Engineering Applications: Modified Alcian Blue Method Development and Validation

1
Department of Chemical Engineering, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka 819-0395, Japan
2
Department of Biochemistry and Laboratory Medicine, School of Biomedicine, Mongolian National University of Medical Sciences, Ulaanbaatar 14210, Mongolia
3
Department of Chemical Engineering & Technology, College of Engineering, Mindanao State University-Iligan Insititute of Technology, Iligan 9200, Philippines
*
Author to whom correspondence should be addressed.
J. Funct. Biomater. 2019, 10(2), 19; https://doi.org/10.3390/jfb10020019
Received: 31 March 2019 / Revised: 25 April 2019 / Accepted: 29 April 2019 / Published: 30 April 2019
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 ℃ 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. View Full-Text
Keywords: sulfated glycosaminoglycans; heparin, alcian blue method; method validation; effect of protein; liver-derived extracellular matrix; tissue engineering sulfated glycosaminoglycans; heparin, alcian blue method; method validation; effect of protein; liver-derived extracellular matrix; tissue engineering
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Iimaa, T.; Ikegami, Y.; Bual, R.; Shirakigawa, N.; Ijima, H. Analysis of Sulfated Glycosaminoglycans in ECM Scaffolds for Tissue Engineering Applications: Modified Alcian Blue Method Development and Validation. J. Funct. Biomater. 2019, 10, 19.

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