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Purpose: The aim of this study was to compare how two innovative laser titanium surfaces and sandblasted and acid-etched surfaces influence human osteoblast behavior during osteogenesis and the initial phases of bone deposition. Materials and Methods: Human osteoblasts from human adipose stem cells were sorted by flow cytometric analysis and induced to differentiate. After 40 days, the osteogenic differentiation was detected by alizarin red staining, and the alkaline phosphatase (ALP) was evaluated with western blot (WB) and real-time reverse transcriptase-polymerase chain reaction (RT-PcR) analysis. After confluence, human osteoblasts were cultured onto two different innovative laser-obtained titanium surfaces (L1 and L2) and compared with one sandblasted and acid-etched (SBAE) surface as the control. At different times, human osteoblast behavior was evaluated with cell proliferation viability assay (MTT), scanning electron microscopy (SEM), energy-dispersive x-rays (EDAX), osteogenic markers with RT-PcR, and WB analysis of matrix extracellular phosphoglycoprotein (MEPE), ALP, and osteocalcin (OCN). Results: After cell sorting, the human osteoblasts from human adipose stem cells showed increasing values of ALP mRNA and protein expression. The osteogenic differentiation was confirmed by quantitative alizarin red staining assay. Profilometric and SEM analysis showed relevant differences between SBAE, L1, and L2 specimens. After 20 days of culture onto titanium samples, SEM evaluation showed a small number of human osteoblasts and isolated sites of bone matrix deposition in SBAE specimens. At the same time, on L1 surfaces, only an osteoblast mono-layer with initial bone deposition was found, while on L2 specimens, there was a thick network with flattened large stellate cells, many cellular interconnections with strong titanium adhesion, and a large complex mineralized structure of crystal bone. After 20 days, for all titanium samples, human osteoblasts culturing EDAX analysis showed the absence of impurities and a higher bone matrix deposition in L2 specimens compared with L1 and SBAE samples. Conclusion: The innovative microtopography and nanotopography laser-induced surface showed high biocompatibility with primary human osteoblast cultures and the absence of impurities. The innovative laser texture was capable of influencing the osteogenic process, confirming the critical role of titanium surface characteristics in the cell adhesion and bone deposition during the early phases of osseointegration. The association of human adipose stem cells and titanium surfaces laser-induced with an innovative procedure could generate promising improvements and developments in orthopedics, maxillofacial, and dental implant surgery. [ABSTRACT FROM AUTHOR]