Biaxial cell stimulation: A mechanical validation.

To analyse mechanotransduction resulting from tensile loading under defined conditions, various devices for in vitro cell stimulation have been developed. This work aimed to determine the strain distribution on the membrane of a commercially available device and its consistency with rising cycle numbers, as well as the amount of strain transferred to adherent cells. The strains and their behaviour within the stimulation device were determined using digital image correlation (DIC). The strain transferred to cells was measured on eGFP-transfected bone marrow-derived cells imaged with a fluorescence microscope. The analysis was performed by determining the coordinates of prominent positions on the cells, calculating vectors between the coordinates and their length changes with increasing applied tensile strain. The stimulation device was found to apply homogeneous (mean of standard deviations approx. 2% of mean strain) and reproducible strains in the central well area. However, on average, only half of the applied strain was transferred to the bone marrow-derived cells. Furthermore, the strain measured within the device increased significantly with an increasing number of cycles while the membrane's Young's modulus decreased, indicating permanent changes in the material during extended use. Thus, strain magnitudes do not match the system readout and results require careful interpretation, especially at high cycle numbers.

Detection of novel skeletogenesis target genes by comprehensive analysis of a Runx2(-/-) mouse model.

Runx2 is an essential factor for skeletogenesis and heterozygous loss causes cleidocranial dysplasia in humans and a corresponding phenotype in the mouse. Homozygous Runx2-deficient mice lack hypertrophic cartilage and bone. We compared the expression profiles of E14.5 wildtype and Runx2(-/-) murine embryonal humeri to identify new transcripts potentially involved in cartilage and bone development. Seventy-one differentially expressed genes were identified by two independent oligonucleotide-microarray hybridizations and quantitative RT-PCR experiments. Gene Ontology analysis demonstrated an enrichment of the differentially regulated genes in annotations to terms such as extracellular, skeletal development, and ossification. In situ hybridization on E15.5 limb sections was performed for all 71 differentially regulated genes. For 54 genes conclusive in situ hybridization results were obtained and all of them showed skeletal expression. Co-expression with Runx2 was demonstrated for 44 genes. While 41 of the 71 differentially expressed genes have a known role in bone and cartilage, we identified 21 known genes that have not yet been implicated in skeletal development and 9 entirely new transcripts. Expression in the developing skeleton was demonstrated for 21 of these genes.