Abstract: Objective To explore the effects of different osteogenic induction methods on the osteogenic differentiation and calcification of human valvular interstitial cells(hVICs). Methods Five commonly used in vitro osteogenic induction methods for valvular interstitial cells were selected from the literature: (1) 2 mmol/L inorganic phosphate+50 μg/mL vitamin C(2 mmol/L Pi + VC); (2) 2.6 mmol/L inorganic phosphate(2.6 mmol/L Pi); (3) 2.5 mmol/L inorganic phosphate+2.7 mmol/L calcium chloride+50 μg/mL vitamin C(2.5 mmol/L Pi + Ca + VC); (4) 10 mmol/L β-glycerophosphate+10 nmol/L dexamethasone+8 mmol/L calcium chloride+4 μg/mL vitamin D3(β-GP+DXM+Ca+VD3); (5) 10 mmol/L β-glycerophosphate+10 nmol/L dexamethasone+50 μg/mL vitamin C(β-GP+DXM+VC). Alizarin red staining, RT-qPCR and Western blotting were used to assess calcium deposition and the expression of osteogenic markers, respectively. Results By comparing the calcium deposition areas on day 4, 7, 10, and 14 induced by five different induction methods, it was found that there was almost no calcification within the first 10 days with 2 mmol/L Pi+VC, β-GP+DXM+Ca+VD3, and β-GP+DXM+VC. In contrast, significant calcification appeared on day 7 with 2.6 mmol/L Pi, 2.5 mmol/L Pi+Ca+VC showed significant calcification on day 4 and almost all calcification on day 7. On day 14, except for the relatively low calcification with 2 mmol/L Pi+VC, all other methods showed significant calcification. Osteogenic marker detection indicated that alkaline phosphatase was significantly upregulated on day 7 with 2 mmol/L Pi+VC, 2.6 mmol/L Pi, and β-GP+DXM+VC(P<0.05), while osteopontin showed significant upregulation on day 7 in all methods(P<0.001). Subsequently, calcium induction was performed with pairwise combinations of the three components in 2.5 mmol/L Pi+Ca+VC. It was found that the calcium deposition areas were significantly larger with Pi+Ca and Pi+Ca+VC on day 7 compared to Pi+VC and Ca+VC, but ALP expression was significantly lower in Pi+Ca and Pi+Ca+VC, suggesting that this calcium deposition might be due to the reaction between inorganic phosphate and calcium chloride forming calcium phosphate precipitates. Conclusion The optimal effect of inducing osteogenic differentiation in hVICs was achieved with 2.6 mmol/L Pi and β-GP+DXM+VC.

Key words: Valvular interstitial cell, Osteogenic induction, Osteogenic differentiation, Calcification, Inorganic phosphate, β-glycerophosphate