Abstract:
Mechanobiology encompasses all of the mechanical processes that influence the cell and
its environment. Thereby, the major actors are the cell´s structural components (i.e. cell
membrane, cytoskeleton), the components of the extracellular matrix (ECM), and the
interface between both (i.e. mechanosensors, adhesion molecules). It is well established
that biomechanics plays a critical role in the homeostasis of living tissue, but the concrete
mechanisms by which mechanical cues are assimilated and regulate the balance between
health and disease are not yet fully understood. In recent decades, atomic force
microscopy (AFM) has emerged as the gold standard to measure the foremost mechanical
marker of a tissue/cell, namely the stiffness (measured as the Young´s modulus). This
parameter provides important information about the state of a living entity, and can be
used to investigate a diseased/degenerative condition. In the present work, AFM was
employed to characterize the biomechanical alterations occurring during two major
disorders from the orthopedic field: osteoarthritis (OA) and bone/soft-tissue cancers.
OA is defined as a degenerative joint condition, characterized by articular cartilage
destruction. With progression of the disease the chondrocyte organization pattern is
evolving, from single string (SS) in healthy cartilage to double string (DS), small cluster
(SC), big cluster (BC) and finally diffuse pattern in the most advanced arthritic state.
Cartilage discs of 1mm thickness and 4mm diameter were generated from condyles of
patient undergoing total knee arthroplasty and were sorted according to the
aforementioned cellular patterns. Subsequently the stiffness of the discs was measured
with AFM. A stepwise stiffness decrease was observed with an increasing degeneration
level, except for the group of the diffuse pattern, which displayed a large data spread with
a relatively high stiffness on average. The discs used for those experiments have the
advantage of being of appreciable thickness, they exhibit the native cartilage surface at
their upmost side and the cells within the tissue are kept alive. They hence represent a
suitable model to further investigate articular cartilage degenerative processes in situ.
Bone and soft-tissue sarcomas represent a group of diverse cancer entities from
mesenchymal origin, with overall poor prognosis. The stiffness of malignant cells of five
different bone and soft-tissue sarcoma cell lines were investigated using AFM and were
compared to their respective healthy control cells. Osteosarcoma, Ewing sarcoma,
fibrosarcoma and rhabdomyosarcoma cells were significantly less stiff than osteoblasts,
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Mesenchymal stem cells (MSCs), fibroblasts and skeletal muscle cells (SKMCs)
respectively, reflecting a typical feature observed in most of the neoplastic
transformation. However, chondrosarcoma cells appeared to be stiffer than chondrocytes
and the other malignant cell lines. Further cytoskeleton examination using fluorescence
microscopy, ELISA and qPCR revealed that if most of the malignant cells displayed a
disorganized internal scaffold and a decreased F-actin content, chondrosarcoma
maintained a cytoskeletal structure relatively close to the one of the healthy cells, with
the microtubule being the most affected components (increase in the β-tubulin gene
expression and protein content). These results define the biomechanical fingerprints of
the investigated cancer cell lines and expose the related molecular mechanisms.
Overall, the work presented in this thesis contributes to the effort of mechanical
characterization of the biological systems respectively addressed in the disclosed studies.
Understanding the processes underlying the biomechanical properties of cells and tissue
is of upmost importance for the development of novel diagnostic and therapy approaches.
