Nanofibers Regulate BMSC Through FAK/YAP/RhoA Pathway

Objectives: Understanding cell-material interaction is a prerequisite for the development of bio-inspired materials for tissue regeneration. The effect of nanofibrous architecture of a biomaterial on stem cell behaviors, however, is largely ambiguous. Current studies of the nanofibrous effect on stem cell fates cannot exclude the interference of cell-cell interaction. This work aims to develop a unique micropatterning biotechnology to single out the influence of cell-cell interaction, and to explore the mechanism of bone marrow stem cell (BMSC) adhesion, proliferation, and differentiation on the nanofibrous architecture.
Methods: An extracellular matrix (ECM)-like nanofibrous gelatin micropattern was developed using a process that combined electrospinning and chemical crosslinking with a polyethylene glycol (PEG)-based photolithography technique. For comparison, a smooth gelatin micropattern was also prepared. Each unit in the nanofibrous or smooth micropattern allows the accommodation of only one rat BMSC. Cellular behaviors including cellular adhesion (spreading speed and focal adhesion formation), cell morphology (shape, area and actin distribution), and osteogenic differentiation (ALP staining) were examined. To verify the role of FAK/YAP/RhoA pathway in linking cell morphology and differentiation on the micropattern, the RhoA/ROCK inhibitor Y27632 was added and the expression and distribution of vinculin and YAP were measured.
Results: The BMSC cultured on the nanofibrous gelatin micropattern exhibited a more in vivo-like morphology that that on smooth micropattern. In addition, the BMSC on the nanofibrous micropattern had a slower spreading speed, smaller spreading area, less focal adhesion formation, less stress fiber and stronger ALP activity. FAK/YAP/RhoA pathways was involved in the process indicated by the difference of the expression and distribution of vinculin and YAP. In addition, the addition of RhoA/ROCK inhibitor Y27632 significantly decrease the osteogenic activity of the BMSC.
Conclusions: Nanofibrous architecture modulates BMSC to present an in vivo-like morphology and enhanced osteogenic activity, and the regulation was involved in the FAK/YAP/RhoA pathway