Dimethylaminobenzoaldehyde

Pellets were harvested after 28 days to measure glycosaminoglycan (GAG), collagen, and DNA content. Samples were digested overnight in a papain digestion buffer (250 μg/mL papain; Sigma-Aldrich, 0.2 M NaH₂PO₄, 0.1M EDTA, 0.01M cysteine, pH 6) at 60°C.
Sulfated GAG content was quantified using a dimethylmethylene blue (DMMB; pH 3) assay. The 525/595 nm absorbance ratio was measured using chondroitin-6-sulfate (Sigma-Aldrich) as a standard.
Total collagen content was calculated from the hydroxyproline content (Neuman and Logan, 1950 (link)). papain digests were lyophilized and hydrolyzed in 4 M NaOH (Sigma-Aldrich) in Milli-Q water overnight at 108°C, after which samples were neutralized with 1.4 M citric acid (Sigma-Aldrich) in Milli-Q water. 50 mM freshly prepared Chloramin-T (Merck) in oxidation buffer was added and incubated for 20 min under agitation. Subsequently, 1.1 M freshly prepared dimethylaminobenzoaldehyde (Merck) in 25% (v/v) perchloric acid (Merck) in 2-propanol (Sigma-Aldrich) was added and incubated for 20 min at 60°C. Samples were cooled and absorbance read at 570 nm using hydroxyproline (Merck) as a standard.
Total DNA content was quantified using a Quant-iT PicoGreen dsDNA assay (Invitrogen) according to the manufacturer’s instructions. Fluorescence was measured at 485 nm excitation and 535 nm emission by a microplate fluorometer (Fluoroskan Ascent).

Pellets were harvested after 28 days and digested using a papain digestion buffer (250 µg/mL papain; Sigma-Aldrich, 0.2 M NaH₂PO₄, 0.1 M ethylenediaminetetraacetic acid [EDTA], 0.01 M cysteine, pH 6) at 60 °C overnight. GAG content in the digests (deposition) and medium (release) was assessed using a dimethylmethylene blue (DMMB; pH 3) assay to quantify sulphated GAGs. The absorbance was measured at 525 and 595 nm using a spectrophotometer and the ratio at 525/595 nm calculated. Chondroitin-6-sulfate (Sigma-Aldrich) was used as a standard.
For the analysis of collagen deposition, digests were lyophilized followed by a hydrolyzation in 4 M NaOH overnight at 108 °C. Samples were neutralized using 1.4 M citric acid. Then, 50 mM chloramine-T (Merck, Darmstadt, Germany) in oxidation buffer was added. After 20 min incubation, dimethylaminobenzoaldehyde (Merck) in 25% (w/v) perchloric acid in 2-propanol was added. Absorbance was measured at 570 nm after 20 min incubation at 60 °C. Hydroxyproline (Merck) was used a standard since 13.5% of collagen is composed of Hydroxyproline [48 (link)]. DNA content of digests was measured using a Quant-iT PicoGreen dsDNA assay (Invitrogen) and was used to normalize collagen and GAG content.