The formation of an ordered surface texture with micro and nanometer features on Ti/Zr multilayers is studied for better understanding and improvement of cell integration. Nanocomposite in form 30×(Ti/Zr)/Si thin films was deposited by ion sputtering on Si substrate for biocompatibility investigation. Surface texturing by femtosecond laser processing made it possible to form the laser-induced periodic surface structure (LIPSS) in each laser-written line. At fluence slightly above the ablation threshold, beside the formation of low spatial frequency-LIPSS (LSFL) oriented perpendicular to the direction of the laser polarization, the laser-induced surface oxidation was achieved on the irradiated area. Intermixing between the Ti and Zr layers with the formation of alloy in the sub-surface region was attained during the laser processing. The surface of the Ti/Zr multilayer system with changed composition and topography was used to observe the effect of topography on the survival, adhesion and proliferation of the murine mesenchymal stem cells (MSCs). Confocal and SEM microscopy images showed that cell adhesion and their growth improve on these modified surfaces, with tendency of the cell orientation along of LIPSS in laser-written lines.Coatings 2019, 9, 854 2 of 12 originates in easy formation of outer Ti-oxide layer with a negative charge at physiological pH and protects against the metallic components dissolving in biological fluids [7]. One promising candidate for alloying is Zr, which act as a neutral element when dissolved in Ti and can improve mechanical properties of alloys [7]. From the biomedical point of view, Zr is fully soluble in both allotropic phases of Ti, improving the mechanical strength, corrosion resistance, and biocompatibility of Ti alloys [8]. Zirconium (Zr) has received special attention as an alloying element in Ti-based alloys, because it acts like non-toxic and non-allergenic elements, thereby avoiding stress shielding effects and implant failure [9]. Binary Ti-Zr alloy has exhibited a good combination of mechanical properties, with the advantage of easy manufacturing, in comparison to multicomponent, Ti-based alloys. Additionally, Zr with the lower elastic modulus (~80 GPa) can contribute to reducing stress shielding, which is the reason for the biomechanical incompatibility between the pure Ti-based implant and the bone [10].Currently, more sophisticated and versatile methods/tools for surface engineering and synthesis of nanoscale facilities for biomedical applications are needed. In traditional chemical and physical methods, although the microstructure can be controlled to some extent, the specific shape and size in some particular applications cannot be precisely controlled. Most of the traditional methods have disadvantages, such as low efficiency, high cost and difficult-to-machine. By contrast, laser processing can easily form and control desired complicated topography with high resolution and high economic efficiency [11,12]. Techniques based on laser surface modification have adva...