Fact versus artifact: avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs.
Fact versus artifact: avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs.
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DOI：
10.22203/ecm.v029a17
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发表时间：
2015-04-19
影响因子：
3.1
通讯作者：
Levenston ME
中科院分区：
文献类型：
--
作者：
Zheng CH;Levenston ME
1，9-二甲基亚甲基蓝（DMMB）测定广泛用于定量工程组织、培养基、组织样品和体液中的硫酸化糖胺聚糖（sGAG）含量，但易受透明质酸（HA）、DNA和RNA等聚阴离子的干扰。我们检查了染料pH和吸光度波长的特定组合是否可以在不影响DMMB测定灵敏度的情况下最小化非sGAG伪影。HA和DNA溶液在pH 3下产生大量信号，但在pH 1.5下不产生。降低染料pH值并没有显著改变正常软骨和半月板组织的sGAG测量值，但消除了酶促分离的软骨细胞、脂肪源性干细胞（ADSC）-琼脂糖构建体和ADSC颗粒的过高的表观sGAG含量。在软骨组织工程案例研究中，pH 3染料表明在基础培养基和软骨形成培养基中的整个培养过程中的高表观sGAG读数，其中软骨形成构建体在第14天和第21天之间显著下降。然而，pH 1.5的染料表明在基础培养基中最小的sGAG积累和在软骨形成培养基中的整个培养过程中稳定的sGAG含量。由于通常难以先验地知道研究中的所有组是否具有足够高的sGAG含量以压倒伪影，因此我们建议修改标准DMMB测定以降低组织工程和临床研究中虚假结果的风险。具体而言，我们建议转换为pH 1.5的DMMB染料，并基于525 nm（μ峰）和595 nm（β峰）之间的吸光度差异进行定量，以补偿与降低染料pH值相关的灵敏度中度损失。
The 1,9-dimethylmethylene blue (DMMB) assay is widely used to quantify sulfated glycosaminoglycan (sGAG) contents of engineered tissues, culture media, tissue samples and bodily fluids, but is subject to interference from polyanions such as hyaluronic acid (HA), DNA and RNA. We examined whether specific combinations of dye pH and absorbance wavelength could minimize non-sGAG artifacts without compromising DMMB assay sensitivity. HA and DNA solutions generated substantial signal at pH 3 but not at pH 1.5. Reducing dye pH did not significantly alter sGAG measurements for normal cartilage and meniscus tissues, but eliminated anomalously high apparent sGAG contents for enzymatically isolated chondrocytes, adipose-derived stem cell (ADSC)-agarose constructs and ADSC pellets. In a cartilage tissue engineering case study, pH 3 dye indicated high apparent sGAG readings throughout culture in both basal and chondrogenic media, with a marked decline between day 14 and 21 for chondrogenic constructs. The pH 1.5 dye, however, indicated minimal sGAG accumulation in basal medium and stable sGAG content throughout culture in chondrogenic medium. As it is often difficult to know a priori whether all groups in a study will have sGAG contents high enough to overwhelm artifacts, we recommend modifying the standard DMMB assay to reduce the risk of spurious findings in tissue engineering and clinical research. Specifically, we recommend shifting to a pH 1.5 DMMB dye and basing quantification on the absorbance difference between 525 nm (μ peak) and 595 nm (β peak) to compensate for the moderate loss of sensitivity associated with reducing the dye pH.
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