Mesenchymal stem cell differentiation on electrochemically modified titanium: an optimized approach for biomedical 
applications.

Purpose: To speed up the osteointegration process, surface-treated titanium has been widely used in dental and orthopedic applications. The present work describes a new silicon-based anodic spark deposition (ASD) treatment and investigates the properties of the surfaces obtained, focusing on their capability to modulate the osteogenic differentiation potential of adult mesenchymal stem cells (MSCs). Methods: The surfaces examined were obtained from commercially pure grade 2 titanium by a single-step ASD (SUM) eventually followed by a thermal treatment in alkali solution (SUMNa), while acid-etched titanium (AE; NextMaterials s.r.l.) was selected as a control. Their morphology, elemental composition, crystallographic structure of the TiO2 layer, wettability and topography were evaluated by scanning electron microscopy, energy-dispersive X-ray spectroscopy, thin-film X-ray diffraction, contact angle measurements and laser profilometry, respectively. MSCs' response to surface properties was assessed by examining cell morphology and viability by scanning electron microscopy and Alamar Blue (TM) assay, while their osteogenic differentiation potential was investigated by evaluating the levels of the enzyme alkaline phosphatase (ALP) and the degree of calcium accumulation by Alizarin Red-S (AR-S) staining. Results: The proposed ASD treatment has allowed the obtaining of surfaces with round-shaped micrometric pores, enriched in calcium, phosphorus and silicon and significantly more wettable than controls; furthermore, the treatment has been shown to promote MSC proliferation and the degree of in vitro mineralization. Conclusions: The described ASD treatment may be an effective technique to modify the surface cues of titanium implants, aiming at enhancing the conveying of osteoprogenitor cells and their functional differentiation in bone cells.