Type I collagen was prepared as described previously 41 (link). Briefly, tendons from rat tails (BioIVT, Westbury, NY) were soaked in 0.1% acetic acid (Kodak, Rochester, NY) for at least 48 hours. The solution was then centrifuged for 90 minutes at 9000 RPM. The supernatant was then collected, frozen, and lyophilized. A stock collagen solution was then created by reconstituting the lyophilized collagen in 0.1% acetic acid at 15 mg/mL.
Pre-glycation was achieved by mixing stock collagen with ribose (Sigma-Aldrich, St. Louis, MO) in 0.1% acetic acid to final ribose concentrations of 0, 125, 250, 375, or 500 mM. After mixing, these solutions were left at 4°C for up to 21 days. ribose was chosen as the reducing sugar for this study due to its higher efficiency in glycating collagen compared to other sugars, such as glucose 35 (link).
To form gels, pre-glycated and control collagen solutions were neutralized with 1X phosphate buffered saline (PBS, Corning cellgro, Manassas, VA), 10X PBS (Corning cellgro, Manassas, VA), and 1N NaOH (Avantor, Center Valley, PA).
These mixtures were then allowed to gel at 37°C before being prepared for FTIR and AGE fluorescence testing. For rheology testing, samples were immediately loaded onto the rheometer after mixing.
Pre-glycation was achieved by mixing stock collagen with ribose (Sigma-Aldrich, St. Louis, MO) in 0.1% acetic acid to final ribose concentrations of 0, 125, 250, 375, or 500 mM. After mixing, these solutions were left at 4°C for up to 21 days. ribose was chosen as the reducing sugar for this study due to its higher efficiency in glycating collagen compared to other sugars, such as glucose 35 (link).
To form gels, pre-glycated and control collagen solutions were neutralized with 1X phosphate buffered saline (PBS, Corning cellgro, Manassas, VA), 10X PBS (Corning cellgro, Manassas, VA), and 1N NaOH (Avantor, Center Valley, PA).
These mixtures were then allowed to gel at 37°C before being prepared for FTIR and AGE fluorescence testing. For rheology testing, samples were immediately loaded onto the rheometer after mixing.
