Strain estimation of fibronectin fibrils using two dimensional digital image correlation

Bone consists of a highly specialized mineralized extracellular matrix (ECM) which supports cells and tissues. Fibronectin is a non-collagenous protein produced by bone cells and is one of the earliest ECM proteins to be assembled. Fibronectin supports the assembly of several other bone matrix proteins, therefore understanding the dynamic process by which the fibronectin is assembled provides insight into how the bone matrix is formed. Live cell imaging of fibronectin assembly in living osteoblasts helps in understanding the kinematics of its assembly, which is a highly dynamic process.;Fibronectin assembly was imaged in 2T3 osteoblasts using time lapse imaging over a 48 hour period. This was divided into four equal 12 hour stages, in which we could observe the assembly process from no fibrils to formation of a mature fibril network. Two Dimensional Digital Image Correlation (2D-DIC), a powerful optical computational tool, was used to quantify the tensile and compressive strains experienced by the assembling fibronectin fibrils due to the extensive cell motion. The 2D-DIC technique was used to quantify the total lengths, large strains and displacements of the assembling fibronectin fibrils. All the code has been developed using Matlab version 12. Seven different movie stacks from two experiments were analyzed. The results quantify the strains experienced by the fibrils at different time stages, due to the underlying cell motion. The average tensile and compression strains were reduced by 6,000microepsilon in the12-24hr movie segment compared to 24-36hr. The average tensile strain was decreased by 6,000 microepsilon and the average compression strain was decreased by 3,000microepsilon in the 24-36hr movie segment compared to 36-48hr. This suggests that fibril strains are reduced as a consequence of matrix maturation. Individual fibrils also showed unique strain profiles, illustrating both the dynamic nature and heterogeneity of fibril motions. This thesis signifies the importance of fibronectin protein in the ECM assembly process by providing strong mathematical and statistical details obtained using the Digital Image Correlation.