Temporal regulation of BMP2 growth factor signaling in response to mechanical loading is linked to cytoskeletal and focal adhesion remodeling.

Biophysical cues have the ability to enhance cellular signaling response to Bone Morphogenetic Proteins, an essential growth factor during bone development and regeneration. Yet, therapeutic application of Bone Morphogenetic Protein 2 (BMP2) is restricted due to uncontrolled side effects. An understanding of the temporal characteristics of mechanically regulated signaling events and underlying mechanism is lacking. Using a 3D bioreactor system in combination with a soft macroporous biomaterial substrate, we mimic the in vivo environment that BMP2 is acting in. We show that the intensity and duration of BMP2 signaling increases with increasing loading frequency in synchrony with the number and size of focal adhesions. Long-term mechanical stimulation increases the expression of BMP receptor type 1B, specific integrin subtypes and integrin clustering. Together, this triggered a short-lived mechanical echo that enhanced BMP2 signaling even when BMP2 is administered directly after mechanical stimulation, but not when it is applied after a resting period of ≥30 min. Interfering with cytoskeletal remodeling hinders focal adhesion remodeling verifying its critical role in shifting cells into a state of high BMP2 responsiveness. The design of biomaterials that exploit this potential locally at the site of injury will help to overcome current limitations of clinical growth factor treatment.

Mechanical heterogeneity in a soft biomaterial niche controls BMP2 signaling.

The extracellular matrix is known to impact cell function during regeneration by modulating growth factor signaling. However, how the mechanical properties and structure of biomaterials can be used to optimize the cellular response to growth factors is widely neglected. Here, we engineered a macroporous biomaterial to study cellular signaling in environments that mimic the mechanical stiffness but also the mechanical heterogeneity of native extracellular matrix. We found that the mechanical interaction of cells with the heterogeneous and non-linear deformation properties of soft matrices (E < 5 kPa) enhances BMP-2 growth factor signaling with high relevance for tissue regeneration. In contrast, this effect is absent in homogeneous hydrogels that are often used to study cell responses to mechanical cues. Live cell imaging and in silico finite element modeling further revealed that a subpopulation of highly active, fast migrating cells is responsible for most of the material deformation, while a second, less active population experiences this deformation as an extrinsic mechanical stimulation. At an overall low cell density, the active cell population dominates the process, suggesting that it plays a particularly important role in early tissue healing scenarios where cells invade tissue defects or implanted biomaterials. Taken together, our findings demonstrate that the mechanical heterogeneity of the natural extracellular matrix environment plays an important role in triggering regeneration by endogenously acting growth factors. This suggests the inclusion of such mechanical complexity as a design parameter in future biomaterials, in addition to established parameters such as mechanical stiffness and stress relaxation.

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.