Passaging effects on mineralization and mRNA expression of rat calvarial cells seeded in monolayer and on 3D polymer constructs in vitro

In this study, primary rat calvarial cells were expanded and then seeded in monolayer and on porous 3D scaffolds to determine the effect of passaging on matrix mineralization and osteogenic gene mRNA production. Passage 1, 2 and 3 cells were seeded in collagen coated 6 well plates and on 5 mm × 3 mm poly(L-lactide-co-D, L-lactide 70:30) (PLDL) discs (n=6 for each group). Monolayer culture was carried out to 3 weeks and plates were stained by Von Kossa to measure mineralization area. Constructs were cultured for 8 weeks and scanned by micro-computed tomography (μCT) at 24 and 57 days to quantify mineralization. Both culture groups were analyzed with real-time quantitative RT-PCR to determine the amount of osteogenic gene mRNA produced. Von Kossa staining showed a statistically significant decrease in the amount of mineralized nodule formation as passage number increased. There was no significant difference in the amount of mineralization produced by each passage group in 3D culture as shown by μCT analysis. There was no reduction in osteogenic mRNA detected by realtime quantitative RT-PCR for osteocalcin (OCN), osteopontin (OPN), osteonectin (ONN) or alkaline phosphatase (ALP) from passage 1 to 3 in 3D culture while 2D culture did show reductions in OCN and OPN with passaging.