RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography Dependent

Abstract

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho-family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA-mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and -mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano-scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity.

Influence of the wettability of different titanium surface topographies on initial cellular behavior

“…Surface topography is recognized as an important factor for better osseointegration and regulation of cellular behavior. Recent studies showed that surface topography modulates some cellular behaviors such as cell morphology, RhoA activation and osteogenic differentiation 7) . Although recent studies have suggested that surface wettability modulated by surface treatments could enhance the early osseointegration process 21) , it remains to be determined how the modulated surface properties reciprocally interact and how they regulate cellular behavior.…”

“…The cells were seeded on D0 and D56 disks at a density of 2.0×10 4 cells/disk and cultured in α-MEM with 3% FBS for 6 h. To visualize cell morphology, the actin filaments were labeled with Texas Red-X phalloidin (Invitrogen, Grand Island, NY, USA) and the nuclei were counterstained with DAPI (Vectashield HardSet Mounting Medium, Thermo Fisher) as described in our previous study 7) . The stained cells were observed under a fluorescence microscope (BZ-9000, Keyence).…”

“…In response to extracellular signals, RhoA induces coordinated changes in the organization of the actin cytoskeleton and in gene transcription to drive a large variety of biological responses including morphogenesis, chemotaxis, axonal guidance, and cell cycle progression 19) . However, these studies, including our previous study 7) , did not take into consideration the effects of surface wettability. Another study showed that the RhoA activity in hydrophobic surface-adherent cells was significantly higher than in cells on hydrophilic surfaces, and that cell adhesion was affected by surface wettability and RhoA activity 20) .…”

“…Surface topography has been reported to improve cellular behavior in some kinds of cells 2,3) . In particular, micrometer-and nanometer-scale surface topographical cues have been known to have the specific capacities to alter cell morphology [4][5][6] in various cells and subsequent cellular behavior 7) . Furthermore, the development of an implant surface with higher wettability is a relatively recent innovation.…”

“…Previous studies indicated that surface wettability could play a critical role in initial cellular characteristics such as cell morphology and cell adhesion 8,9) . Surface topography, including microstructured and nanostructured surfaces, has also been known to modulate cell morphology and the activity of RhoA, a small GTPase protein of the Rho family, in cytoskeletal regulation 7,18) . RhoA is known to act as a molecular switch.…”