Trace metal grade concentrated hno3

Inductively Coupled Plasma (ICP) Optical Emission spectrometry was performed to assess mineral formation at the end of in vitro culture (day 28). MC, MC-HB, and MC-Fluffy samples were washed in PBS, fixed with formalin (Formal-Fix, Thermo Fisher Scientific) for 24 hrs at 4°C, washed again in PBS, then dried on a Kimwipe before storing at −80°C until use. Before performing ICP, samples were lyophilized at the same conditions used to fabricate the original scaffolds. Samples were weighed then dissolved using concentrated nitric acid, Trace Metal Grade concentrated HNO₃ (Thermo Fischer Scientific 67–70%), followed by automated sequential microwave digestion in a CEM Mars 6 microwave digester. The acidic solution was diluted to a volume of 50 mL using DI water, so as to make the final concentration of the acid <5%. The ICP-OES was calibrated with a series of matrix matched standards before introducing the unknown samples. Digestion and ICP-OES analysis parameters are listed in Supp. Tables 4 and 5. Nine samples were used for each group and these were normalized to the calcium and phosphorous content of respective dry scaffolds and composites without cells in order to get a fold change and new calcium and phosphorous deposition.

Calcium and Phosphate release from 3D-printed Hyperelastic Bone and mineralized collagen scaffolds were quantified through a 28 days standard release experiment (culture in 5 mL of PBS at 37°C). 1 mL PBS aliquots were removed from suspension, then combined with 2 mL of Trace Metal Grade concentrated HNO₃ (Thermo Fisher Scientific 67–70%) for a 30-minute pre-digestion step. The tubes were then capped and placed into a rotating carousel inside MARS 6 (CEM Microwave Technology Ltd., North Carolina, USA) microwave digester (40 minutes). The final digested solution was diluted to a volume of 50 mL in DI water, then analyzed via inductively coupled plasma-mass spectrometer (ICP-OES, Optima 8300, PerkinElmer, USA; elemental analysis in axial mode; Supp. Tables 1 and 2). Eight samples of each group were used per timepoint.