Evaluation of bone formation in neonatal mouse calvariae using micro-CT and histomorphometry: an in vitro study

Neonatal calvarial bone has been widely used for investigating the biological behaviour of intramembranous bones. This work evaluated the bone formation of neonatal calvarial bone by microcomputed tomography (micro-CT) and histomorphometry. Moreover, the viability of neonatal calvarial bone and the effect of micro-CT radiation exposure on neonatal calvarial bone viability were investigated. The calvarial bones of 4-day-old CD-1 mice were cultured in Dulbecco's modified Eagle's medium (DMEM) or osteogenic medium (OM) for 23 days. Micro-CT scanning and histological analysis were performed on days 2, 9, 16 and 23. An "OM-control" group was scanned only on days 2 and 23 to evaluate the effect of a single micro-CT radiation dose on calvarial bones. Histomorphometric measurements revealed that the number of osteoblasts per unit bone surface area (N. Ob/BS, /mm²) (days 9, 16 and 23) and the number of osteoclasts per unit bone surface area (N. Oc/BS, /mm²) (days 9 and 16) were higher and lower, respectively, in the OM group than in the DMEM group. Moreover, the calvarial bone survived for at least 16 days in vitro, as indicated by tartrate-resistant acid phosphatase (TRAP)-positive staining. Micro-CT assessment revealed that the bone surface (BS), bone volume (BV), bone surface density (BS/TV(Tissue volume)) and percent bone volume (BV/TV) were greater in the OM group than in the DMEM group except at baseline on day 2. All bone parameters of calvariae cultured in OM and OM-control conditions were not significantly different on days 2 and 23. Thus, the radiation dose from micro-CT in our study design had no perceptible effect on the formation of mouse calvarial bone in vitro.