Introduction:The ambition of current bone biomaterials research is to designimplants that induce controlled, guided, successful and rapid healing. Tiatnium and its alloys are most widely used biomaterials in hardtissue surgeries.Numerous studies have proved that the osteoblastic cells are betteradapted to a nanoscale environment rather to microscale niche.Nanoporous materials are in the focus of scientific queries due to theirexclusive properties and very broad area of applications. One of suchmaterial is nanostructured anodic titanium oxide (ATO) with highlyordered, vertically aligned nanotubes. It is hypothesized thatnanotopography of TiO2 nanotubes more closely mirror the porousstructure of native bone enhancing optimal interactions for contactosteogenesis. Yet, the best nanopore size have not been fully exploredand established.Stem cells are regarded as very promising and inviting tool for tissueengineering and cell-based therapies. Adipose derived stem cells(ASCs) offer many interesting opportunities for regenerativemedicine. They represent many advantages over other mesenchymalprogenitors (large deposits, easier, safer and less invasive accessibility,established isolation procedure, etc.)The important goal of tissue engineering is to direct stem cellsdifferentiation into a specific, desired cell lineage.Aim:The aim of this study was to assess cell response, growth, viability,metabolic activity and ability to differentiate into osteoblastic lineageof human ASCs from subcutaneous fat tissue on nanoporous TiO2(with the pore diameters of 80 and 108 nm).Methods:A three-step electrochemical anodization process in glycerol-basedelectrolyte with fluoride ions was performed to produce a nanoporousoxide coat on Ti surfaces. Surface characterization of thenanostructured oxide layer and cell morphology was carried out withelectron scanning microscopy. Human adipose derived stem cells wereisolated from subcutaneous fat tissue of healthy donors and culturedon titanium samples for 21 days. In vitro experiments to assess cellgrowth (BrdU incorporation test), activity (ATP monitoring and totalprotein concentration) and viability (LDH release test) were carriedout. Cell differentiation was determined by alkaline phosphataseactivity, gene expression (BGLAP, SP7, TBX3, DLX5, RUNX2,ALPL, BMP2K, COL1A1, SPP1, NOTCH1, SPARC) and alizarin redstaining.Results:A nanoporous layer of TiO2 was successfully produced on Ti surfacesusing a relatively simple and economic technique. The nanostructuredoxide coat significantly induced the osteo-differentiation of ASCsafter 21-day in vitro culture in comparison with conventional titanium.There were no powerful differences between two nanoporous (80 nmvs 108 nm) surfaces.Conclusion:Our results indicate that anodic nanostructured TiO2 is safe andnontoxic biomaterial. Moreover it can induce osteo-differentiation ofhuman ASCs from abdominal subcutaneous fat tissue in vitro culture.The potential benefit from these studies would be extremely useful inthe fundamental understanding of cell biology and in the applicationof new modifications for biomaterials and implant design.Further in vitro and in vivo test should be performed to assess ASCabasedtreatment strategies and tissue regeneration potential of implants opsonized with autologous stem cells.