Growth factor effects on single chondrocyte biomechanics and gene expression

摘要

Studying chondrocyte responses to mechanical forces and how growth factors modify these responses allows for exploration of the underlying principles of cartilage physiology and disease. The work described in this thesis aims to understand single chondrocyte response to mechanical stimulation and how soluble growth factors can modulate this response. This knowledge is valuable for the formulation of successful cartilage tissue repair and replacement strategies, as well as etiopathogenesis of and treatments for the disease osteoarthritis. The first portion of this thesis established unconfined compression as a method for creep testing single adherent chondrocytes. Fitting of three continuum biomechanics models showed that a continuum viscolelastic model best described the creep behavior of single chondrocytes. Unconfined creep compression was next used to test single superficial and middle/deep zone chondrocytes. The effects of growth factor treatment (TGF-beta1, IGF-I, and TGF-beta1 + IGF-I) and seeding time (3 and 18 hr) were also tested. Creep testing demonstrated that all growth factor treatments stiffened the cytoskeleton of single cells, and staining showed increased F-actin due to growth factors. Techniques were developed to isolate specific adherent single chondrocytes and assay their gene expression with real-time RT-PCR. These techniques were used to detect significant differences in the gene expression of single zonal chondrocytes exposed to IGF-I. Finally, single cells were statically compressed with or without TGF-beta1 and IGF-I and their resulting gene expression was measured. Static compression elicited catabolic gene expression in control single cells. TGF-beta1 and IGF-I provided mechanoprotection and differentially prevented this catabolic response. Cytoimmunohistochemistry of single chondrocytes fixed in compression demonstrated that nearly all axial strain experienced by the cell is experienced by the nucleus. Also, cells compressed at higher levels of force had increasingly deformed nuclei with larger spaces in the chromatin. These results suggest that transcription is modified directly by nuclear strains through force-mediated changes of the chromatin. This work provides the first evidence of mechanical forces modifying gene expression and provides a starting point for future studies where precise thresholds of mechanical stimulation required to elicit desired metabolic changes in single cells will be determined.