Osteogenic induction in avian adult aortic valve interstitial cells (AVICs) in vitro. (a, b) Representative bright-field images of uninduced control (a, marked by arrowheads) and osteogenic (OS) media-induced AVICs in culture (b, marked by arrowheads). (c) Gene expressions of osteogenic markers (Osteopontin, Runx2, ALP, Msx2, and Osterix), by reverse-transcriptase PCR, have shown elevated expression in OS-induced AVICs compared to control normal AVICs. β-Actin was used for equal mRNA input and RT efficiency. (d) (I–V): relative mRNA level expressions have been further validated by real-time PCR. Osteopontin, Runx2, ALP, Msx2, and Osterix all increased by 3.65 fold, 2.68 fold, 0.4 fold, 2.68 fold, and 17.12 fold, respectively. β-Actin was used to analyze normalized gene expressions. Statistical significance was determined by Student's t-test, where ∗ denotes p ≤ 0.05 and n = 3.