Gene expression profiles of dynamically compressed single chondrocytes and chondrons
ABSTRACT A chondrocyte produces a hydrated pericellular matrix (PCM); together they form a chondron. Previous work has shown that the presence of the PCM influences the biological response of chondrocytes to loading. The objective of this study was to determine the gene expression profiles of enzymatically isolated single chondrocytes and chondrons in response to dynamic compression. Cartilage specific extracellular matrix components and transcription factors were examined. Following dynamic compression, chondrocytes and chondrons showed variations in gene expression profiles. Aggrecan, Type II collagen and osteopontin gene expression were significantly increased in chondrons. Lubricin gene expression decreased in both chondrons and chondrocytes. Dynamic compression had no effect on SOX9 gene expression. Our results demonstrate a clear role for the PCM in interfacing the mechanical signalling in chondrocytes in response to dynamic compression. Further investigation of single chondrocytes and chondrons from different zones within articular cartilage may further our understanding of cartilage mechanobiology.
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ABSTRACT: One approach widely used in tissue engineering research, particularly for compressive load-bearing tissues, has been to embed progenitor cells in a hydrogel scaffold. Because these cells are exposed to mechanical loading either during culture or when implanted, understanding the biophysical mechanisms that regulate cell deformation in these systems could help enhance the efficiency of construct maturation. This study investigates the role of the pericellular matrix (PCM) in the deformability of human mesenchymal stem cells (hMSCs) in hydrogels during chondrogenesis. Results show that type VI collagen accumulated regionally around cells after 1week and fully enveloped cells by 2weeks of chondrogenic alginate bead culture, but was minimally deposited around cells in growth media. The PCM was able to attenuate cell deformations significantly only when type VI collagen coverage was complete. Gene expression data show that sox9 was upregulated when non-differentiating cells were embedded in alginate, but not sustained unless supplemented with pro-chondrogenic factors. In addition, we examined cell deformations with disruption of individual cytoskeletal elements, but could not detect significant effects. Overall, these finding suggest that the PCM is the primary modulator of cell deformation during chondrogenic differentiation of hMSCs. These considerations will be important for defining loading strategies and designing scaffold properties. KeywordsPericellular matrix-Cytoskeleton-Cell deformation-Mesenchymal stem cells-Gene expressionCellular and Molecular Bioengineering 12/2010; 3(4):387-397. · 1.23 Impact Factor
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ABSTRACT: The aim of this study was to explore how cell-matrix interactions and extrinsic mechanical signals interact to determine stem cell fate in response to transforming growth factor-β3 (TGF-β3). Bone marrow derived mesenchymal stem cells (MSCs) were seeded in agarose and fibrin hydrogels and subjected to dynamic compression in the presence of different concentrations of TGF-β3. Markers of chondrogenic, myogenic and endochondral differentiation were assessed. MSCs embedded within agarose hydrogels adopted a spherical cell morphology, while cells directly adhered to the fibrin matrix and took on a spread morphology. Free-swelling agarose constructs stained positively for chondrogenic markers, with MSCs appearing to progress towards terminal differentiation as indicated by mineral staining. MSC seeded fibrin constructs progressed along an alternative myogenic pathway in long-term free-swelling culture. Dynamic compression suppressed differentiation towards any investigated lineage in both fibrin and agarose hydrogels in the short-term. Given that fibrin clots have been shown to support a chondrogenic phenotype in vivo within mechanically loaded joint defect environments, we next explored the influence of long term (42 days) dynamic compression on MSC differentiation. Mechanical signals generated by this extrinsic loading ultimately governed MSC fate, directing MSCs along a chondrogenic pathway as opposed to the default myogenic phenotype supported within unloaded fibrin clots. In conclusion, this study demonstrates that external cues such as the mechanical environment can override the influence specific substrates, scaffolds or hydrogels have on determining mesenchymal stem cell fate. The temporal data presented in this study highlights the importance of considering how MSCs respond to extrinsic mechanical signals in the long term.Journal of biomechanics 08/2012; 45(15):2483-92. · 2.66 Impact Factor
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ABSTRACT: It remains unclear how specific mechanical signals generated by applied dynamic compression (DC) regulate chondrocyte biosynthetic activity. It has previously been suggested that DC-induced interstitial fluid flow positively impacts cartilage-specific matrix production. Modifying fluid flow within dynamically compressed hydrogels therefore represents a promising approach to controlling chondrocyte behavior, which could potentially be achieved by changing the construct architecture. The objective of this study was to first determine the influence of construct architecture on the mechanical environment within dynamically compressed agarose hydrogels using finite element (FE) modeling and to then investigate how chondrocytes would respond to this altered environment. To modify construct architecture, an array of channels was introduced into the hydrogels. Increased magnitudes of fluid flow were predicted in the periphery of dynamically compressed solid hydrogels and also around the channels in the dynamically compressed channeled hydrogels. DC was found to significantly increase sGAG synthesis in solid constructs, which could be attributed at least in part to an increase in DNA. DC was also found to preferentially increase collagen accumulation in regions of solid and channeled constructs where FE modeling predicted higher levels of fluid flow, suggesting that this stimulus is important for promoting collagen production by chondrocytes embedded in agarose gels. In conclusion, this study demonstrates how the architecture of cell-seeded scaffolds or hydrogels can be modified to alter the spatial levels of biophysical cues throughout the construct, leading to greater collagen accumulation throughout the engineered tissue rather than preferentially in the construct periphery. This system also provides a novel approach to investigate how chondrocytes respond to altered levels of biophysical stimulation.Biomechanics and Modeling in Mechanobiology 11/2012; · 3.33 Impact Factor