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<p>The role of articular cartilage in diarthrodial joints is primarily mechanical
as the tissue provides a nearly frictionless, load-bearing surface that supports and
distributes forces generated during joint loading. Embedded within the extensive cartilage
extracellular matrix (ECM), chondrocytes are surrounded by a narrow, distinct pericellular
matrix (PCM) that is thought to regulate the biomechanical microenvironment of the
cell and influence chondrocyte metabolism, cartilage homeostasis, and overall joint
health. While previous studies of PCM mechanical properties required physical extraction
of the cell and PCM from the tissue, atomic force microscopy (AFM) provides a means
for high resolution microindentation testing that can be used to measure local mechanical
properties in situ. This dissertation develops and applies AFM microindentation techniques
to 1) evaluate the microscale elastic properties of the cartilage PCM and ECM in situ
and 2) correlate site-specific biochemical composition with biomechanical properties
of the PCM and ECM. </p><p>An AFM-based stiffness mapping technique was experimentally
validated and applied to cartilage sections to evaluate ECM and PCM properties in
situ with minimal disruption of native matrix integration. As expected, PCM elastic
moduli were significantly less than ECM moduli, uniform with depth, and mechanically
isotropic. ECM moduli exhibited distinct depth-dependent anisotropy and unexpectedly,
were found to decrease with depth from the articular surface. Both the PCM and ECM
exhibited alterations in microscale moduli and their spatial distributions when evaluated
in cartilage presenting early degenerative changes associated with osteoarthritis
(OA) as compared to healthy tissue. </p><p>The ability to correlate site-specific
biochemical composition with local biomechanical properties provides a more complete
characterization of the chondrocyte microenvironment. To this end, we developed novel
immunofluorescence (IF)-guided AFM stiffness mapping and demonstrated that PCM mechanical
properties correlate with the presence of type VI collagen. Extending this technique
by using dual IF, we presented new evidence for a defining role of perlecan in the
PCM, showing that interior regions of the PCM rich in perlecan and type VI collagen
exhibit lower elastic moduli than peripheral PCM and ECM regions lacking perlecan.
Furthermore, lower moduli at the PCM interior were significantly influenced by the
presence of heparan sulfate. IF-guided AFM stiffness mapping was combined with enzymatic
digestion to demonstrate that the micromechanical properties of the PCM exhibit high
resistance to specific enzymatic digestion of aggrecan and aggrecan-associated glycosaminoglycans
but are vulnerable to proteolytic degradation by leukocyte elastase. </p><p>Overall,
this research generates new insights into the complex structural, compositional, and
functional relationships between the cartilage ECM and PCM and provides the tools
and framework for further studies to continue to investigate their importance in regulating
chondrocyte physiology in health and disease.</p>
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