Pericellular matrix mechanotransduction events in differentiating human mesenchymal stem cells: Modulating the pericellular matrix through silencing type VI collagen and decorin
Twomey, Julianne Doreen
Hsieh, Adam H
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Stem cell therapies are currently being explored for their potential in the regeneration of load bearing tissues, such as cartilage. Current therapies lack the ability to intrinsically overcome a mechanically adverse environment at implantation. To advance the implementation of human mesenchymal stem cells (hMSCs) for cartilage repair, the mechanisms by which cells “feel“ and interact with their micromechanical environment need to be understood. Chondrogenic hMSCs develop a thin pericellular matrix (PCM), consisting of type VI collagen (ColVI) and proteoglycans such as decorin (DCN). The PCM is believed to control mechanotransduction events, acting as both a biomechanical and biochemical buffer. This thesis studies the functional role of ColVI and DCN through targeted gene knockdown using shRNA lentiviral vectors complimentary to <italic>col6a1</italic> or <italic>dcn</italic>. In the first part of the work, the biophysical role of the PCM was determined through comparisons of cellular deformability under uniaxial strain with or without ColVI and DCN knockdown. HMSCs were cultured in alginate scaffolds and were stimulated with transforming growth factor β for 1 to 2 weeks. We found that the PCM with ColVI knockdown lacked the ability to withstand applied compression and with DCN knockdown deformed in a strain– dependent manner. Next we analyzed the mechanosignaling initiation caused by a transient sinusoidal compressive load through studying cytoskeletal kinetics and gene expression. Altering the PCM through ColVI and DCN knockdown caused an increase in actin and vimentin cytoskeletal protein concentration that lacked a dynamic response to load. This lead to a stronger fibroblast growth factor gene expression in ColVI knockdown. DCN also demonstrated direct control over cartilage oligomeric matrix protein gene expression, through a loss of TGF– β regulation. These results were further demonstrated during long term compressive culture. Unconfined sinusoidal compressive culture revealed the highest improvement in material properties in knockdown samples at day 14. Through these studies, we demonstrated that ColVI and DCN are integral proteins in maintaining the structural microenvironment through protecting the cell from injurious deformation, maintaining cytoskeletal dynamics in response to load, and regulating the differentiation rate through TGF– β signaling. Finally, we demonstrated the ability to manipulate chondrogenic mechanotransduction events using genetic engineering.