Characterization of mineralized bone trabeculae formed in vitro by the OmGFP66 osteogenic cell line
Benazir Khurshida, Aisha Mousab, Joseph Deeringb, and Marc McKeeb,c
a-Bioengineering, b-Dental Medicine & Oral Health Sciences, c-anatomy & Cell Biology, 平特五不中, Montreal
Knowing how bone cells regulate mineralization is important to understanding skeletal development. To date, few cell culture systems reliably recapitulate skeletal events as they occur in vivo. Recently, a mouse cell line named OmGFP66 was developed1 that produces mineralized structures that resemble bone trabeculae, and they have both surface osteoblasts and matrix/mineral-embedded internal osteocytes constitutively expressing bone cell marker genes. Together, these two cell types generate a characteristic
lacunocanalicular network housing the osteocytes and their connected dendritic extensions within the trabeculae. Here, we characterize the cell culture in 3D using X-ray microscopy (submicron micro-computed tomography) and focused ion beam-scanning electron microscopy (FIB-SEM). PCR of RNA extracts showed osteogenic marker gene expression for Phex, Spp1 (Opn), E11/gp38, Sost, Fgf23, mDmp1, Enpp1, and Mepe, confirming their bone cell phenotype. Histology of the trabeculae revealed elongated mineralized trabeculae that were further characterized by X-ray microscopy, elemental EDS microprobe mapping, and FIB-SEM tomography. Examination of 257 trabeculae by X-ray microscopy revealed osteocytes completely embedded within distinct, fully formed mineral lacunae (closed) and at the forming surface as partial mineral lacuna (open). Individual trabeculae had volumes ranging from 700 碌m3 to 600,000 碌m3 with a median volume of 14,200 碌m3 . The width and length dimensions of the trabeculae were heterogeneous, having a median of 27卤17 碌m and 59卤46 碌m, respectively, and a median aspect ratio of 3.7. Of 2,297 segmented osteocyte mineral lacunae, 38% were of the closed phenotype, and these had a median volume of 347 碌m3 , similar to the value of 405卤37 渭m3 observed in mice2 . X-ray microscopy observations at the microscale were correlated with nanoscale characterizations by FIB-SEM tomography. In conclusion, we provide 3D quantitative volumetric data for the OmGFP66 cell line that in vitro remarkably produces bone-like mineralized trabeculae with a resident osteocyte lacunocanalicular network similar to bone.
1Wang, K et al J Bone Miner Res 34:979 (2019), 2Hoac B et al J Bone Miner Res 35:2032 (2020)