Osteogenesis RT2 Profiler PCR Array

Osteogenesis PCR array was performed, in triplicate for each biological sample, in hBMSC cultures grown on the biomaterial in order to identify genes from the osteogenic pathway activated by the scaffold. Specifically, total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen, Milan, Italy) (Mazzoni et al., 2020) according to the manufacturer’s instructions from cells grown on (i) Coll/Pro Osteon^® 200 scaffolding and (ii) TCPS (control group) (Manfrini et al., 2013). RNA was quantified using a NanoDrop spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, DE, United States) (Mazzoni et al., 2017, 2020). The Human Osteogenesis RT² Profiler PCR Array (Qiagen, Milan, Italy) was used as described (Mazzoni et al., 2020). Specific primer sets employed in real-time PCRs were used to analyze the expression of 84 genes involved in different pathways, such as osteogenic differentiation, cartilage condensation, ossification, bone metabolism, bone mineralization, binding to Ca²⁺ and homeostasis, extracellular matrix (ECM) protease inhibitors, adhesion molecules, cell-to-cell adhesion, ECM adhesion molecules, and growth factors. All reactions were performed in triplicate. For data analysis, the fold change (FC) of each gene expression was calculated using the 2^–ΔΔCt method, whereas housekeeping genes, employed as controls, were used to normalize results and Log₂ FC; < 1 or >1 was considered significant (Mazzoni et al., 2017).