Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro.

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16Ink4a or p21Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.

Characterizing cell recruitment into isotropic and anisotropic biomaterials by quantification of spatial density gradients in vitro.

The success of cell-free in situ tissue engineering approaches depends on an appropriate recruitment of autologous cells from neighboring tissues. This identifies cellular migration as a critical parameter for the pre-clinical characterization of biomaterials. Here, we present a new method to quantify both the extent and the spatial anisotropy of cell migration in vitro. For this purpose, a cell spheroid is used as a cell source to provide a high number of cells for cellular invasion and, at the same time, to guarantee a controlled and spatially localized contact to the material. Therefore, current limitations of assays based on 2D cell sources can be overcome. We tested the method on three biomaterials that are in clinical use for soft tissue augmentation in maxilla-facial surgery and a substrate used for 3D in vitro cell culture. The selected biomaterials were all collagen-derived, but differed in their internal architecture. The analysis of cellular isodensity profiles within the biomaterials allowed the identification of the extent and the preferential directions of migration, as well as their relation to the biomaterials and their specific pore morphologies. The higher cell density within the biomaterials resulting from the here-introduced cell spheroid assay compared to established 2D cell layer assays suggests a better representation of the in vivo situation. Consequently, the presented method is proposed to advance the pre-clinical evaluation of cell recruitment into biomaterials, possibly leading to an improved prediction of the regeneration outcome.