Hyaluronic Acid Impact on Osteogenic Differentiation of SaOS-2-Cells in Airlift Model

Objectives: Gene expression profile of SAOS-2 cells seeded on two different collagen substrates for osteogenic differentiation alone or stimulated additionally by hyaluronic acid in three concentrations was studied at two time points.
Methods: Substrates were placed on semipermeable membranes of airlift-inserts mounted into 6-well plates lightly touching medium surface. SAOS-2 cells were seeded in organoid order on both, one collagen substrate cross-linked by nature, one by ribose (NCLC, RCLC). Negative control (NC) contained medium alone, Positive Control (PC) used osteogenic differentiation medium (β-glycerophosphate and ascorbic acid). Substrates in test groups were hydrated by cross-linked hyaluronic acid (HA) in 1:100 (C1), 1:10 (C2) and 1:1 (C3) dilutions as adjunctive to osteogenic differentiation medium. After nourishing period of one (T1) and two (T2) weeks cells were sampled by scraper. RUNX2, BGLAP, IBSP, Connexin43 (Cx43), and Periostin expression was analyzed by RT-qPCR using SYBR green®. All experiments were triplicated. ANOVA statistics and multi-variance analyses used △△-CT scores, calculated in relation to NC and PC after adjusting expression levels of targets to housekeeping GAPDH, respectively (SPSS 23.0, USA).
Results: Periostin had to be excluded from analysis due to high heterogeneity in data and missing values. Total expression of other targets showed substrate and time dependent profile. Delta-delta CT related to PC showed P=0.000 for culture duration, P=0.028 for substrate and P=0.022 for culture and group. NC revealed 1.569 to 1.309-fold and 3.864 to 1.405- fold total expressions at T1 and T2, respectively, both favoring RCLC vs. NCLC substrates. Stimulation in C1 resulted in significantly higher rates on NCLC vs. RCLC (3.826 vs. 1.191-fold; P=0.032) at T1; in C2 level of significance was underrun (P=0.052). At T2 differences were non-significant; values in C1 to C3 were higher for NCLC vs. RCLC substrates. Rates for single targets will be presented.
Conclusions: These preliminary data demonstrated efficient nourishing of cells via diffusion. Using NCLC or RCLC substrates gene transcribing activity of SAOS-2 cells was maintained at T1 and T2 with tendency to decline at the latter in NC. Stimulation by HA had impact on expression rates in cross-over manner, favoring NCLC vs. RCLC at T1 and T2.