Using In Vitro Culture to Detect Changes in Bone Matrix Mechanical Properties - 7. PROJECT SUMMARY The goal of this Exploratory/Developmental project is to develop a way of assessing mechanical failure of bone matrix using the same material used to evaluate bone formation in vitro. Mechanical failure (fracture) of bone is the primary clinical concern in bone disease. Pharmaceutical interventions to prevent mechanical failure of bone focus on increasing bone size or density (i.e. bone quantity and BMD). These approaches are successful, but their long-term use either has diminishing returns and/or increased risk of adverse effects. The mechanical properties of bone extracellular matrix are a major contributor to bone fragility but are only indirectly addressed by existing therapeutics. A major roadblock to identifying modifiable factors that influence bone matrix is the effort required for early-stage discovery of factors influencing bone matrix: Screening for factors that enhance bone formation can be performed using cell modified and cultured in vitro (weeks of effort). In contrast, detecting a change in mechanical failure of bone matrix (fracture toughness, strength) requires specimens as large as whole mouse bones, necessitating the development of a transgenic mouse (months of effort). To overcome this challenge, we propose using micropillars (~5-10 um diameter) generated by focused ion beams to evaluate mechanical failure in bone matrix in bony nodules generated by in vitro culture. Encouraged by PRELIMINARY STUDIES demonstrating high repeatability of micropillars in bone and the feasibility of generating micropillars from bony nodules, here we establish the micropillar in the context of deficiency of the heterotrimeric G protein Gs in mice, a condition that results in severe bone fragility. The project has one aim: determine the effects of deficiency of GS on the fracture toughness of bone matrix using bony nodules formed in vitro and bone matrix formed in vivo and includes mechanical testing of bony nodules generated by precursor cells in which GS is removed in vitro, bone matrix from the GS deficient mouse and osteoprogenitors reprogrammed from human fibroblasts in which GS is removed in vitro. In addition to addressing the rate limiting factor to identifying factors that influence bone matrix mechanical properties, the proposed technique will have broad applications including localized measures of matrix strength at localized areas including within a fracture callus or the peri-implant space.
