Quantification of Sulfated Glycosaminoglycans in Chondrocyte/Alginate Cultures, by Use of 1,9-Dimethylmethylene Blue

Enobakhare, B.; Bader, Dan L.; Lee, David A.

doi:10.1006/abio.1996.0502

Cited by 290 publications

(199 citation statements)

References 3 publications

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“…The 1,9- dimethylmethylene blue (DMMB) assay was conducted to measure the amount of sGAG in hydrogel construct. Shark chondroitin sulfate (Sigma, MO) was used as the standard of sGAG amount under the DMMB assay 53,54 . The hydroxyproline assay was applied to measure the amount of collagen contents in gels after acid hydrolysis 55 .…”

Section: Methodsmentioning

confidence: 99%

Mechanical confinement regulates cartilage matrix formation by chondrocytes

et al. 2017

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Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for 3D culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1β, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

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Section: Methodsmentioning

confidence: 99%

Mechanical confinement regulates cartilage matrix formation by chondrocytes

et al. 2017

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“…The release of glycosaminoglycans from the meniscal explants, as a measure of proteoglycan degradation, was determined using the dimethylmethylene blue (DMMB) method as described previously (13,16). Media samples were added to prepared DMMB solution, and sulfated glycosaminoglycan levels were determined spectrophotometrically with absorbance read at 570 nm.…”

Section: Methodsmentioning

confidence: 99%

Regulation of matrix turnover in meniscal explants: role of mechanical stress, interleukin-1, and nitric oxide

Shin¹,

Fermor²,

Weinberg³

et al. 2003

Journal of Applied Physiology

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. Regulation of matrix turnover in meniscal explants: role of mechanical stress, interleukin-1, and nitric oxide. J Appl Physiol 95: 308-313, 2003. First published March 28, 2003 10.1152/ japplphysiol.00131.2003The meniscus is an intra-articular fibrocartilaginous structure that serves essential biomechanical roles in the knee. With injury or arthritis, the meniscus may be exposed to significant changes in its biochemical and biomechanical environments that likely contribute to the progression of joint disease. The goal of this study was to examine the influence of mechanical stress on matrix turnover in the meniscus in the presence of interleukin-1 (IL-1) and to determine the role of nitric oxide (NO) in these processes. Explants of porcine menisci were subjected to dynamic compressive stresses at 0.1 MPa for 24 h at 0.5 Hz with 1 ng/ml IL-1, and the synthesis of total protein, proteoglycan, and NO was measured. The effects of a nitric oxide synthase 2 (NOS2) inhibitor were determined. Dynamic compression significantly increased protein and proteoglycan synthesis by 68 and 58%, respectively, compared with uncompressed explants. This stimulatory effect of mechanical stress was prevented by the presence of IL-1 but was restored by specifically inhibiting NOS2. Release of proteoglycans into the medium was increased by IL-1 or mechanical compression and further enhanced by IL-1 and compression together. Stimulation of proteoglycan release in response to compression was dependent on NOS2 regardless of the presence of IL-1. These finding suggest that IL-1 may modulate the effects of mechanical stress on extracellular matrix turnover through a pathway that is dependent on NO.

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“…Samples were weighed, refrozen, lyophilized, and weighed again. Samples were then digested with 1.25 mg/mL papain (Sigma-Aldrich) solution overnight at 60°C and analyzed for DNA content through the Hoechst DNA assay, 33 sulfated glycosaminoglycan (GAG) content through a modified 1,9-dimethylmethylene blue assay, 34 and collagen through a hydroxyproline assay. 35 Biochemical properties are reported normalized to the dry weight of the samples.…”

Section: Biochemical Analysesmentioning

confidence: 99%