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: 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
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ABSTRACT: Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.Histochemie 06/2011; 136(2):153-62. · 2.61 Impact Factor