Objectives Aortic valve interstitial cells (AVICs) play a vital role in the development of aortic valve stenosis. The aim of this study is to observe that whether or not AVICs calcified spontaneously and to detect the expression of osteogenic factors in the formation of calcified cell nodules.
Methods AVICs were cultured in explant method, calcification were detected by Von Kossa, alizarin red S stain and a calcium assay kit, and expression of osteogenic factors, such as Cbfa1/ OC/BMP-2/BMP-4, were detected by RT-PCR and western-blot analysis at different times.
AVICs aggregated and formed small cell nodules after 4∼6 day culture, and the number of nodules become more and the diameter is bigger at day 12 than that at day 6.
The cell nodules were positive by Von Kossa and alizarin red S stain, which indicated that they were calcified nodules. The statistical analysis indicated that both the content of alizarin red S and calcium at day 12 were higher than that at day 0 and day 6 (F = 106.167, F = 116.379, respectively; P<0.001).
The statistic analysis of the osteogenic gene expression indicated that the Cbfa1/OC/ BMP- 4 mRNA were lowest at day 0, higher at day 6 and highest at day 12 (F = 410.106, X2 = 7.200, F = 38.463, respectively; P < 0.005), and BMP-2 mRNA were lowest at day 0, higher at day 6 and day 12 (F = 56.565, P < 0.001). The statistic analysis of the osteogenic protein expression indicated that the BMP-2/4 protein expression were lowest at day 0, higher at day 6 and highest at day 12 (x2 = 7.200, F = 452.482, respectively; P < 0.05).
Conclusions AVICs calcified spontaneously with extend culture time, in which the differentiation of AVICs into osteoblasts-like cells may play an important role in the process of calcification.