The aim of this study was to investigate the impact of cross-linked hyaluronic acid on osteoblast-like cells seeded on top of two collagen substrates, native porcine pericardium membrane (substrate A) and ribose cross-linked collagen membranes (substrate B), in an air-lift model. Substrates A or B, saturated with three hyaluronic acid concentrations, served as membranes for SAOS-2 cells seeded on top. Cultivation followed for 7 and 14 days in the air-lift model. Controls used the same substrates without hyaluronic pre-treatment. Cells were harvested, and four (Runx2, BGLAP, IBSP, Cx43) different osteogenic differentiation markers were assessed by qPCR. Triplicated experiment outcomes were statistically analyzed (ANOVA, t-test; SPSS). Supplementary histologic analysis confirmed the cells' vitality. After seven days, only few markers were overexpressed on both substrates. After 14 days, targeted genes were highly expressed on substrate A. The same substrate treated with 1:100 diluted xHyA disclosed statistically significant different expression level vs. substrate B (p = 0.032). Time (p = 0.0001), experimental condition as a function of time (p = 0.022), and substrate (p = 0.028) were statistically significant factors. Histological imaging demonstrated vitality and visualized nuclei. We conclude that the impact of hyaluronic acid resulted in a higher expression profile of SAOS-2 cells on substrate A compared to substrate B in an air-lift culture after two weeks.
Keywords: SAOS-2 cells; air-lift organoid model; cross-linked collagen substrate; cross-linked hyaluronic acid; osteogenic differentiation.