Abstract
Summary
Attachment, proliferation and osteogenic maturation of hMSCs are enhanced on a sub-micron grooved Ti6Al4V alloy, while osteoblasts are less sensitive. These effects are attributed to their different maturation stage and may be mediated through differential activation of the RhoA/ROCK pathway.
Introduction
Ti6Al4V alloy is the most widely used titanium-based biomaterial for manufacturing bone-anchoring devices. We report on the interactions of human bone-forming cells, mesenchymal stem cells from bone marrow (hMSCs) and primary osteoblasts (hOBs), with an anisotropic Ti6Al4V alloy that displays submicron grooves.
Materials
Submicrometric Ti6Al4V surfaces (GV) with parallel grooves and mean average roughness values around 200 nm were generated by mechanical abrasion. A polished Ti6Al4V surface (PL) was used for comparative purposes. hMSCs were phenotypically characterised as progenitors and hOBs as committed osteogenic cells at a late stage of maturation. Cell attachment, proliferation, cytoskeleton organisation, adhesion sites and fibronectin matrix distribution, RhoA and ROCK distributions and activities, and osteogenic maturation, were analysed in cells cultured on the investigated alloys by means of scanning electron and confocal microscopy and biochemical assays.
Results
hMSCs adhered and proliferated at a higher rate on GV alloy than on PL, while no differences were found in hOBs behavior. Patterned Ti6Al4V alloy promoted the orientation of hOBs and hMSCs in the direction of the anisotropy. Filopodia were involved in the alignment of both cell types along the grinding direction while cells exhibited a lamellipodia-dominated state on PL samples. In both cell types, focal adhesions, actin and tubulin networks and fibronectin extracellular matrix aligned with the direction of the grooves. RhoA/ROCK signaling contributed to hMSCs alignment on the patterned alloy, while osteoblastic orientation does not rely on the activation of this pathway. RhoA activity increased in both cell types cultured on GV alloy and interference on RhoA functioning by treatment with C3 transferase led to loss of organisation of actin and tubulin cytoskeletons. ROCK activity of hMSCs was up-regulated on GV samples, but not affected in hOBs. Treatment with hydroxyfasudil, an inhibitor of ROCK activity, disrupted the alignment of adhesion sites in hMSCs but not in hOBs. RhoA/ROCK signaling is not involved in the orientation of microtubule bundles of hOBs neither hMSCs on any surface, suggesting that such process is controlled by the RhoA effector mDia. When cultured on media that support osteogenic maturation, hMSCs and hOBs behaved differently on the anisotropic surfaces. OPN secretion increased in hMSCs cultured on GV alloy while it remained unaffected in hOBs. Cell mineralisation proceeded to a same extent in hMSCs cultured on the two metallic surfaces but decreased in hOBs cultured on the patterned samples.
Discussion/Conclusion
Collected results indicate that hOBs are less sensitive to the patterned topography of Ti6Al4V alloy than hMSCs. These effects could be attributed to their different stages of maturation and can be mediated, at least in part, through ROCK since its activity increased on hMSCs cultured on the patterned alloy, while hOBs failed to up-regulate it. Collectively, these findings indicate that topography of Ti6Al4V can be manipulated to control cell functions via contact guidance responses. Altogether with the available data in the literature, results herein may also provide guidance to the implant technology for the design of Ti-based alloys with bioinspired surfaces topographies able to enhance specific bone cell activities.
