The pericellular matrix (PCM) is a voluminous polymer network adhered to and surrounding many different types of mammalian cells, and which extends out into the environment outside the cell for distances ranging up to twenty microns. It is comprised of very long flexible polymers (hyaluronan) which are tethered to the cell surface and which have binding sites for large, highly charged bottle brush proteoglycans (aggrecan). The PCM plays an important role in many cell functions such as cell proliferation, cell adhesion, cell migration, and cancer development, however the precise way it influences these processes remains unclear. Three original biophysical tools are developed in this thesis in order to study the PCM: the quantitative particle exclusion assay (qPEA), optical force probe assay (OFPA), and exogenous fluorescent aggrecan mapping assays. These tools are used to measure the polymeric and biophysical properties of the matrix in order to make further advancements in the understanding the PCMs role in adhesion, transport to and from the cell surface, its purported function as a chemical micro-reservoir, as well as basic studies on the kinetics of its formation, turnover and maintenance. The qPEAs measure the penetration and distribution of sub-micron particles after they diffuse into the cell coat, where their distribution maps the interior structure of the PCM. The qPEA assays reveal that the PCM acts a sieve, separating incoming particles by their size, preventing micron sized particles from entering the PCM while allowing sub 100 nm particles to pass to the cell surface. The OFPA uses an optically-trapped bead to study the force response of the matrix as it encounters the probe. The assay not only reveals new details about the PCM such as the fact that it is larger than initially thought, having a two layer structure, but when combined with a polymer physics model which relates the observed equilibrium forces to an existing osmotic pressure gradient within the PCM, the OFPA studies produce the first discovery and measurement of the correlation length distribution in the cell coat. The OFPA and qPEA assays are also performed on cells modified with exogenous aggrecan, resulting in a model for possible proteoglycan mediated cell coat transformations. The fluorescent exogenous aggrecan assays measure the dynamics of the exogenous aggrecan binding to and releasing from the coat, revealing that the PCM can be rapidly modified by a changing environment, and quantitatively measure how the exogenous aggrecan modifies the existing PCM. Together, these assays provide an unprecedented look into the interior structure of the PCM, and the mechanisms responsible both for this structure and its modification.

Articular cartilage is the connective tissue that lines joints and provides a smooth surface for articulation and shock absorption. Osteoarthritis, the progressive degeneration of cartilage, is a painful, debilitating, and widespread disease, affecting 70% of people over 65. Because cartilage is avascular, molecular transport occurs primarily via diffusion. The goal of these studies was to examine whether cartilage matrix structure and composition have a significant effect on diffusive transport. We hypothesized that diffusion is anisotropic in the surface zone of cartilage where collagen structure is aligned and densely packed. A theoretical model and experimental protocol for fluorescence imaging of continuous point photobleaching (FICOPP) were developed to measure diffusional anisotropy. Significant anisotropy was observed in ligament, a highly ordered collagenous tissue. In less ordered articular cartilage, diffusional anisotropy was dependent on site in the tissue and size of the diffusing molecule. These findings suggest that diffusional transport of macromolecules is anisotropic in collagenous tissues, with higher rates of diffusion along primary orientation of collagen fibers. We hypothesized that structural differences in the pericellular matrix of cartilage (PCM) would lead to differences in diffusive properties as compared to the surrounding extracellular matrix (ECM). We modified the scanning microphotolysis (SCAMP) technique to allow measurement of diffusion coefficients within the PCM. Diffusion coefficients in the PCM were lower than in the adjacent ECM in normal cartilage, but with early stage arthritis, the PCM diffusivity was not different from that of the ECM. These data suggest that breakdown of the PCM is an early step in arthritis development. We hypothesized that compression of cartilage would cause site‐specific diffusivity decreases and diffusional anisotropy increases. We utilized SCAMP and FICOPP to measure diffusion coefficients and diffusional anisotropy in cartilage as it was compressed. We found that diffusivity decreased and anisotropy increased with increasing strain in a site‐specific manner. These findings suggest that the high surface zone strains that lead to low diffusivity and high anisotropy will decrease transport between cartilage and synovial fluid in compressed cartilage. We have shown that matrix structure and composition have a significant effect on diffusive transport in cartilage. / Dissertation