Quantifying Hydrogel Matrix Deposition

After 0, 3, 7, 14, 28, or 35 days of culture, biochemical assays were used to assess cell viability by DNA content (PicoGreen assay), cartilage-like matrix synthesis by sGAG content (DMMB assay), and bone-like matrix deposition by Ca²⁺ content (Arsenazo III assay).^{17 (link),18 (link)} All data were normalized to acellular controls cultured under the same conditions and then expressed relative to hydrogel wet mass.^{16 (link)}Hydrogels were washed in PBS for 15 min at 37 °C and transferred to sterilized, pre-weighed polystyrene tubes, each containing a 5 mm stainless steel bead. Each hydrogel was weighed to determine its wet mass and then frozen at −20 °C until ready for characterization. Thawed samples were homogenized in 500 μL of sterile ultrapure H₂O using a Qiagen TissueLyser II (Hilden, Germany) at 30 s⁻¹ for 5 min. A 60 μL aliquot of each suspension was then mixed in 1:1 volume ratio with 1M acetic acid and incubated at room temperature for 16 hr to free Ca²⁺ ions for quantification using the Arsenazo III assay kit.^{17 (link)} The remaining 440 μL of homogenized sample suspension was then mixed in 1:1 volume ratio with an established digestion buffer containing 2 mg/mL proteinase K, 20 μg/mL pepstatin A, and 370 μg/mL iodoacetamide in tris–EDTA solution (12.11 mg/mL tris(hydroxymethyl aminomethane), 0.744 mg/mL EDTA, pH 7.6) to free DNA and sGAG content, and allowed to digest at 60 °C for 16 hr.^{18 (link)} The DNA and sGAG content of the digested samples were then quantified using PicoGreen and DMMB assay kits, respectively.