Bone Abstracts

It is hypothesized that osteocytes regulate bone adaptation by sensing mechanical strains in their microenvironments and signaling net bone formation or resorption. Owing to bone’s anisotropic architecture, individual osteocytes within a bone experience varying strains under mechanical loading. Thus, to accurately determine the relationships between mechanical strain, osteocyte behavior, and bone remodeling, it is crucial to use a single-cell approach. Using an in vivo model for bone adaptation and in vivo μCT, we can register time-lapsed images of bone and quantify regions of formation and resorption. Furthermore, using laser capture microdissection techniques, we can isolate single cells for subsequent gene expression analysis. Mapping of these individual gene expression profiles back to their original locations in the co-registered μCT volumes will therefore greatly enhance our understanding of osteocyte behavior in vivo. To move towards a transcriptome wide analysis of single osteocytes, we have developed a protocol which is able to quantify gene expression in small groups of osteocytes. Female C57BL/6 mice vertebrae were cryosectioned (12 μm thickness), stained in 1% Cresyl Violet in 75% ethanol, dehydrated in an ethanol gradient, and microdissected using a P.A.L.M. laser microscope into PCR tube caps. A two-step Taqman qRT-PCR protocol was used for gene expression analysis. Lysis, RT, and pre-amplification were performed using the CellsDirect One-Step qRT-PCR kit. The resulting cDNA was diluted and analyzed using a standard Taqman qPCR protocol. Our results show that we can detect expression of HPRT-1 in three cells (Ct=24.8±0.2) and ten cells (CT=23.1±0.8). These results prove the feasibility of gene expression analysis of individually microdissected osteocytes. Lab-on-a-chip applications are now being developed to enhance sensitivity and permit the analysis of hundreds of molecular targets.