The experiments proposed in this revised grant application represent the application of an RT-PCR approach to determine mRNA copy number from cultures of rat calvarial osteoblast cells. The rationale for this approach is to provide a means to analyze small numbers of nontransformed cells in a rapid, quantifiable manner. One of our ultimate goals is to adapt the approaches utilized in this proposal to the evaluation of various responses of adult human-derived material. Clinical biopsy material is often quantitative limited and in vitro, the bone-like phenotype is stable for only one or two passages. The RT-PCR assay coupled with the use of internal standards, provides a unique means to evaluate subtle changes in message levels that could not be observed with conventional methods. Since oral bone loss is a significant clinical problem, understanding changes in steady state message for matrix related proteins and important developmental genes upon exposure to membrane deformation may lead to defining mechanisms underlying this pathologic state. The goal of this project is to validate and apply a competitive RT-PCR method for quantitation of changes in bone noncollagenous mRNA copy number in response to physiological parameters of mechanical strain. This goal will be achieved by the following specific aims: [SA 1] Develop a library of bone matrix-related mRNA probes for quantitating copy number. Efficiency and fidelity of cDNA formation will be evaluated along with formation of specific deletion mutations (""""""""midgets"""""""") to be coamplified as PCR standards to quantitate copy number in the cDNA pool. [SA 2] Determine changes in mRNA copy number for bone matrix-related genes in response to physiological (magnitude, frequency and cycles) of strain. Ultimately we wish to identify those aspects of strain-induced bone cell responses associated with changes in message levels.
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| Stanford, C M; Solursh, M; Keller, J C (1999) Significant role of adhesion properties of primary osteoblast-like cells in early adhesion events for chondroitin sulfate and dermatan sulfate surface molecules. J Biomed Mater Res 47:345-52 |
