Search

Physicochemical features of a cell nanoenvironment exert important influence on stem cell behavior and include the influence of matrix elasticity and topography on differentiation processes. The presence of growth factors such as TGF-β and BMPs on these matrices provides chemical cues and thus plays vital role in directing eventual stem cell fate. Engineering of functional biomimetic scaffolds that present programmed spatio-temporal physical and chemical signals to stem cells holds great promise in stem cell therapy. Progress in this field requires tacit understanding of the mechanistic aspects of cell-environment nanointeractions, so that they can be manipulated and exploited for the design of sophisticated next generation biomaterials. In this review, we report and discuss the evolution of these processes and pathways in the context of matrix adhesion as they might relate to stemness and stem cell differentiation. Super-resolution microscopy and single-molecule methods for in vitro nano-manipulation are helping to identify and characterize the molecules and mechanics of structural transitions within stem cells and matrices. All these advances facilitate research toward understanding of stem cell niche and consequently to developing new class of biomaterials helping the “used biomaterials” for applications in tissue engineering and regenerative medicine.

The reflection of picosecond ultrasonic pulses from a cell-substrate interface is used to probe cell-biomaterial adhesion with a subcell resolution. We culture monocytes on top of a thin biocompatible Ti metal film, supported by a transparent sapphire substrate. Low-energy femtosecond pump laser pulses are focused at the bottom of the Ti film to a micron spot. The subsequent ultrafast thermal expansion launches a longitudinal acoustic pulse in Ti, with a broad spectrum extending up to 100 GHz. We measure the acoustic echoes reflected from the Ti-cell interface through the transient optical reflectance changes. The time-frequency analysis of the reflected acoustic pulses gives access to a map of the cell acoustic impedance Zc and to a map of the film-cell interfacial stiffness K simultaneously. Variations in Zc across the cell are attributed to rigidity and density fluctuations within the cell, whereas variations in K are related to interfacial intermolecular forces and to the nano-architecture of the transmembrane bonds.

Different studies have established a link between adult mesenchymal stem cells (MSCs) and perivascular cells, such as pericytes, which envelop the vascular tubes. This link was validated mainly through shared marker signatures and raised the hypothesis that all MSCs are pericytes. A controversy remains, however, as to whether there are functions specifi c to only one cell type. A specifi c role of pericytes in vascular tube stabilization that is not shared with MSCs is reported. An experimental model is introduced for the induction of endothelial cell (EC) tubulogenesis after 24 h of incubation on micropatterned polymer surfaces. Pericytes or MSCs are added separately to this system to evaluate their effect on tubular stabilization. In the absence of additional cells or in the presence only of MSCs the tubular structures are lost after 36 h. Addition of only pericytes, however, stabilizes the EC vasculogenic tubes. Furthermore, only pericytes, but not MSCs, are able to migrate through a mimetic basement membrane and interact with ECs to stabilize lumen structures. It is suggested that pericytes have specifi c cell functions of potential major importance in vascular and bone-tissue regeneration. The use of pericytes in tissue engineering will thus become critical. Blood vessels are composed of two interacting cell types

International audience; Microenvironments such as protein composition, physical features, geometry, and elasticity play important roles in stem cell lineage specification. The components of the extracellular matrix are known to subsequently assemble into fibrillar networks in vivo with defined periodicity. However, the effect of the most critical parameter, which involves the periodicity of these fibrillar networks, on the stem cell fate is not yet investigated. Here, we show the effect of synthetic fibrillar networks patterned with nanometric periodicities, using bottom-up approaches, on the response of stem cells. We have used helical organic nanoribbons based on self-assemblies of Gemini-type amphiphiles to access chiral silica nanoribbons with two different shapes and periodicities (twisted ribbons and helical ribbons) from the same native self-assembled organic nanostructure. We demonstrate the covalent grafting of these silica nanoribbons onto activated glass substrates and the influence of this programmed isotropically oriented matrix to direct the commitment of human mesenchymal stem cells (hMSCs) into osteoblast lineage in vitro, free of osteogenic-inducing media. The specific periodicity of 63 nm (±5 nm) with helical ribbon shape induces specific cell adhesion through the fibrillar focal adhesion formation and leads to stem cell commitment into osteoblast lineage. In contrast, the matrix of periodicity 100 nm (±15 nm) with twisted ribbon shape does not lead to osteoblast commitment. The inhibition of non-muscle myosin II with blebbistatin is sufficient to block this osteoblast commitment on helical nanoribbon matrix, demonstrating that stem cells interpret the nanohelical shape and periodicity environment physically. These results indicate that hMSCs could interpret nanohelical shape and periodicity in the same way they sense microenvironment elasticity. This provides a promising tool to promote hMSC osteogenic capacity, which can be exploited in a 3D scaffold for bone tissue engineering.

Item does not contain fulltext Bulk matrix stiffness has emerged as a key mechanical cue in stem cell differentiation. Here, we show that the commitment and differentiation of human mesenchymal stem cells encapsulated in physiologically soft ( approximately 0.2-0.4 kPa), fully synthetic polyisocyanopeptide-based three-dimensional (3D) matrices that mimic the stiffness of adult stem cell niches and show biopolymer-like stress stiffening, can be readily switched from adipogenesis to osteogenesis by changing only the onset of stress stiffening. This mechanical behaviour can be tuned by simply altering the material's polymer length whilst maintaining stiffness and ligand density. Our findings introduce stress stiffening as an important parameter that governs stem cell fate in a 3D microenvironment, and reveal a correlation between the onset of stiffening and the expression of the microtubule-associated protein DCAMKL1, thus implicating DCAMKL1 in a stress-stiffening-mediated, mechanotransduction pathway that involves microtubule dynamics in stem cell osteogenesis.

Aim: The aim of the present work was to investigate ultrafast laser surface texturing as a surface treatment of Ti-6Al-4V alloy dental and orthopedic implants to improve osteoblastic commitment of human mesenchymal stem cells (hMSCs). Materials & methods: Surface texturing was carried out by direct writing with an Yb:KYW chirped-pulse regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. The surface topography and chemical composition were investigated by scanning electron microscopy and x-ray photoelectron spectroscopy, respectively. Three types of surface textures with potential interest to improve implant osseointegration can be produced by this method: laser-induced periodic surface structures (LIPSSs); nanopillars (NPs); and microcolumns covered with LIPSSs, forming a bimodal roughness distribution. The potential of the laser treatment in improving hMSC differentiation was assessed by in vitro study of hMSCs spreading, adhesion, elongation and differentiation using epifluorescence microscopy at different times after cell seeding, after specific stainings and immunostainings. Results: Cell area and focal adhesion area were lower on the laser-textured surfaces than on a polished reference surface. Obviously, the laser-textured surfaces have an impact on cell shape. Osteoblastic commitment was observed independently of the surface topography after 2 weeks of cell seeding. When the cells were cultured (after 4 weeks of seeding) in osteogenic medium, LIPSS- and NP- textured surfaces enhanced matrix mineralization and bone-like nodule formation as compared with polished and microcolumn-textured surfaces. Conclusion: The present work shows that surface nanotextures consisting of LIPSSs and NPs can, potentially, improve hMSC differentiation into an osteoblastic lineage.

The mechanical properties of cells play a key role in several fundamental biological processes, such as migration, proliferation, differentiation and tissue morphogenesis. The complexity of the inner cell composition and the intricate meshwork formed by transmembrane cell-substrate interactions demands a non-invasive technique to probe cell mechanics and cell adhesion at a subcell scale. In this paper we review the use of laser-generated GHz acoustic waves—a technique called picosecond ultrasonics (PU)—to probe the mechanical properties of single cells. We first describe applications to vegetal cells and biomimetic systems. We show how these systems can be used as simple models to understand more complex animal cells. We then present an opto-acoustic bio-transducer designed for in vivo measurements in physiological conditions. We illustrate the use of this transducer through the simultaneous probing of the density and compressibility of Allium cepa cells. Finally, we demonstrate that this technique can quantify animal-cell adhesion on metallic surfaces by analyzing the acoustic pulses reflected off the cell-metal interface. This innovative approach allows investigating quantitatively cell mechanics without fluorescent labels or mechanical contact to the cell.

The interior of the cell nucleus is comparable to a solid network bathed in an interstitial fluid. From the extrapolation of low frequency data, it is expected that such network should dictate the response of the nucleus to mechanical stress at high frequencies, described by unique elastic moduli. However, none of the existing techniques that can probe the mechanical properties of cells can exceed the kHz range, and the mechanics of the nuclear network remain poorly understood. We use laser-generated acoustic waves to probe remotely the stiffness and viscosity of nuclei in single cells in the previously unexplored GHz range with a ∼100 nm axial resolution. The probing of cells at contrasted differentiation stages, ranging from stem cells to mature cells originating from different tissues, demonstrates that the mechanical properties of the nuclear network are common across various cell types. This points to an asymptotically increasing influence of a solid meshwork of connected chromatin fibers.

Several aspects of the extracellular matrix (ECM) microenvironment, such as protein composition, topographical features and elasticity, have been reported to play critical roles in stem cell lineage specification. In vivo, the ECM is composed of nano-fibrillar networks of defined periodicity. Changes in the periodicity and shape of this nanofiber matrix have been implicated in the induction of several diseases. Despite this, till date, there has been no investigation of the effect of the nanoperiodicity of the ECM nanostructure on the fate of stem cells. In this report, we use helical organic nanoribbons based on self-assembled Gemini amphiphiles to access chiral silica nanoribbons with different shapes (twisted/helical) and periodicity. Glass substrates covalently grafted with ‘RGD’ functionalized helical nanoribbons of periodicity 63 nm induced specific hMSC adhesion through fibrillar focal contact formation, and commitment towards osteoblast lineage in absence of osteogenic-inducing media. In contrast, the substrate with twisted nanoribbons of periodicity 100 nm failed to induce osteogenic commitment. Inhibition of non-muscle myosin II with blebbistatin was sufficient to block osteogenic commitment on helical nanoribbon matrix, demonstrating that the stem cells interpreted nanohelical shape and periodicity environment in the same way they sense microenvironment elasticity. This study thus provides a promising tool to promote osteogenic capacity and may find applications in bone tissue engineering. silica nanostructures, nanohelical periodicity, stem cell microenvironment, cell differentiation.

Biomaterials aim to mimic in vivo extracellular matrices where cell interactions occur on the nanoscale. Thus, incorporation of nanosized components is interesting in the preparation of bioactive surfaces. We present a technique using nanoimprint lithography to create chemical nanopatterns on silicon surfaces functionalized with bioactive motifs. Due to high throughput and versatility, a wide range of geometries and dimensions can be efficiently patterned. In our study, we prepared and characterized two types of bioactive nanodots (150 nm diameter with 350 nm spacing, and 80 nm diameter with 110 nm spacing) functionalized with cell adhesion-promoting RGD peptides. We examined mesenchymal stem cell adhesion and commitment on these modified material surfaces with respect to homogeneous RGD and non-functionalized surfaces. We report that bioactive nanostructures induce fibrillar adhesions on human mesenchymal stem cells with an impact on their behavior and dynamics specifically in terms of cell spreading, cell-material contact, and cell differentiation.

We present a method of preparing and characterizing nanostructured bioactive motifs using a combination of nanoimprint lithography and surface functionalization. Nanodots were fabricated on silicon surfaces and modified with a cell-adhesive RGD peptide for studies in human mesenchymal stem cell adhesion and differentiation. We report that bioactive nanostructures induce mature focal adhesions on human mesenchymal stem cells with an impact on their behavior and dynamics specifically in terms of cell spreading, cell-material contact, and cell differentiation.

Summary Mature osteoblasts are the cells responsible for bone formation and are derived from precursor osteoblasts. However, the mechanisms that control this differentiation are poorly understood. In fact, unlike the majority of organs in the body, which are composed of “soft” tissue from which cells can easily be isolated and studied, the “hard” mineralized tissue of bone has made it difficult to study the function of bone cells. Here, we established an in vitro model that mimics this differentiation under physiological conditions. We obtained mature osteoblasts and characterized them on the basis of the following parameters: the strong expression of osteoblastic markers, such as Runx2 and Col-I; the achievement of specific dimensions (the cell volume increases 26-fold compared to the osteoblast precursors); and the production of an abundant extracellular matrix also called osteoid. We demonstrated that the differentiation of osteoblast precursors into mature osteoblasts requires the continuous activation of Bone Morphogenetic Protein (BMP) receptors, which we established with the immobilization of a BMP-2mimetic peptide on a synthetic matrix mimicking in vivo microenvironment. Importantly, we demonstrated that the organization of the F-actin network and acetylated microtubules of the cells were modified during the differentiation process. We showed that the perturbation of the F-actin cytoskeleton organization abolished the differentiation process. In addition, we demonstrated that expression of the Runx2 gene is required for this differentiation. These findings demonstrate the retro-regulation of cytoplasmic and genic components due to the continuous induction of BMP-2 and also provide more detailed insights into the correct signaling of BMPs for cell differentiation in bone tissue.

H ow endothelial cells (ECs) express the particular fi lopodial or lamellipodial form of the actin machinery is critical to understanding EC functions such as angiogenesis and sprouting. It is not known how these mechanisms coordinately promote lumen formation of ECs. Here, adhesion molecules (RGD peptides) and inductor molecules (BMP-2 mimetic peptides) are micropatterned onto polymer surfaces by a photolithographic technique to induce fi lopodial and lamellipodial migration modes. Firstly, the effects of peptide microgeometrical distribution on EC adhesion, orientation and morphogenesis are evaluated. Large micropatterns (100 μ m) promote EC orientation without lumen formation, whereas small micropatterns (10‐50 μ m) elicit a collective cell organization and induce EC lumen formation, in the case of RGD peptides. Secondly, the correlation between EC actin machinery expression and EC self-assembly into lumen formation is addressed. Only the fi lopodial migration mode (mimicked by RGD) but not lamellipodial migration mode (mimicked by BMP-2) promotes EC lumen formation. This work gives a new concept for the design of biomaterials for tissue engineering and may provide new insight for angiogenesis inhibition on tumors.

Stem cells cultured on extracellular matrix (ECM) with different stiffnesses have been shown to engage into different lineage commitments. However, in vivo, the components of the ECM are known to bind and strongly interact with growth factors. The effect, on the stem cell fate, of the cooperation between the mechanical properties and the growth factor in the same microenvironment has not yet been investigated. Here, we propose a protocol for mimicking this stem cell microenvironment with an in vitro system. This system consists in grafting (without using a spacer) biomolecules that contain N-termini groups onto hydrogel (poly(acrylamide-co-acrylic acid)) surfaces of various stiffnesses ranging from 0.5 to 70 kPa. First, we demonstrate that the commitment of mesenchymal stem cell populations changes in response to the substrate's rigidity, with myogenic differentiation occurring at 13-17 kPa and osteogenic differentiation at 45-49 kPa. Chemical grafting of soft and stiff matrices with an osteogenic factor (BMP-2(mimetic peptide)) results only in osteogenic differentiation. Also, when grafted on even softer gels (0.5-3.5 kPa), the BMP-2(mimetic peptide) had no effect on the stem cell differentiation. We prove that correct organization of F-actin cytoskeleton due to the mechanical properties of the microenvironment is necessary for BMP-induced smad1/5/8 phosphorylation and nuclear translocation. These results suggest that stem cell differentiation is dictated mechanically, but in the presence of a biochemical factor, the effect of the mechanical factor on stem cell commitment is modified. This can explain the diversity of stem cell behaviors in vivo where different growth factors are sequestrated on the ECM.

International audience; This work reports on the synthesis of titanium bone implants functionalized with nanoparticles (NPs) containing Arg-Gly-Asp-Cys peptide (RGDC) and shows the adhesion behavior of cells seeded on these materials. RGDC peptides were first: conjugated to a norbornenyl-poly(ethylene oxide) macromonomer (Nb-PEO). Then, functional NPs with a size of similar to 300 nm and constituted of polynorbornene core surrounded by poly(ethylene oxide) shell were prepared by ring-opening metathesis polymerization in dispersed medium. The grafting density of these NPs on the titanium surface is up to 2 NPs.mu m(-2) (80 pmol of RGDC per cm(-2) of NP surface). Cell adhesion was evaluated using preosteoblast cells (MC3T3-E1). Results of cells cultured for 24 h showed that materials grafted with NPs functionalized with RGDC peptides enhance specific cell adhesion and can create filopodia-like among NP sites by stressing the cells.

International audience; The differentiation of stem cells can be modulated by physical factors such as the micro- and nano-topography of the extracellular matrix. One important goal in stem cell research is to understand the concept that directs differentiation into a specific cell lineage in the nanoscale environment. Here, we demonstrate that such paths exist by controlling only the micro- and nano-topography of polymer surfaces. Altering the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts, in the absence of osteogenic medium. Small (10 nm) depth patterns promoted cell adhesion without noticeable differentiation, whereas larger depth patterns (100 nm) elicited a collective cell organization, which induced selective differentiation into osteoblast-like cells. This latter response was dictated by stress through focal-adhesion-induced reorganization of F-actin filaments. The results have significant implications for understanding the architectural effects of the in vivo microenvironment and also for the therapeutic use of stem cells.

Angiogenesis, the formation of new blood vessels by sprouting from pre-existing ones, is critical for the establishment and maintenance of complex tissues. Angiogenesis is usually triggered by soluble growth factors such as VEGF. However, geometrical cues also play an important role in this process. Here we report the induction of angiogenesis solely by SVVYGLR peptide micropatterning on polymer surfaces. SVVYGLR peptide stripes were micropatterned onto polymer surfaces by photolithography to study their effects on endothelial cell (EC) behavior. Our results showed that the EC behaviors (cell spreading, orientation and migration) were significantly more guided and regulated on narrower SVVYGLR micropatterns (10 and 50 µm) than on larger stripes (100 µm). Also, EC morphogenesis into tube formation was switched on onto the smaller patterns. We illustrated that the central lumen of tubular structures can be formed by only one-to-four cells due to geometrical constraints on the micropatterns which mediated cell-substrate adhesion and generated a correct maturation of adherens junctions. In addition, sprouting of ECs and vascular networks were also induced by geometrical cues on surfaces micropatterned with SVVYGLR peptides. These micropatterned surfaces provide opportunities for mimicking angiogenesis by peptide modification instead of exogenous growth factors. The organization of ECs into tubular structures and the induction of sprouting angiogenesis are important towards the fabrication of vascularized tissues, and this work has great potential applications in tissue engineering and tissue regeneration.

We use femtosecond laser pulses absorbed in a metallic transducer, namely the picosecond ultrasonics technique, for the remote optical generation and detection of GHz acoustic frequencies in single cells by pump-probe sampling. Samples are MC3T3 cells adhering on a TiAl4V alloy substrate. Both pump and probe beams are focused at the cell/transducer interface. The pump absorption yields a temperature rise in the absorbing substrate and a picosecond acoustic pulse is generated through the thermoelastic effect. The probe beam is partially reflected from the metallic interface and partially scattered by the acoustic wavefront propagating in the transparent cell. The change of reflectivity of the cell is measured as a function of the pump-probe time delay. Interferences arise from the two probe contributions causing the so-called Brillouin oscillations. Optical phase variations due to acoustic-induced changes in cell thickness are simultaneously measured. The result of a semi-analytical calculation is fitted to the experimental data. Acoustic frequencies are detected at 30 GHz in the nucleus of single osteoblast cells.

Probing the mechanical properties of the nucleus is essential to understanding whole-cell mechanics under different physiological conditions. The nucleus has a complex composition yielding intricate rheological behavior that is appealing for measurements over a wide frequency range. However none of the existing techniques can exceed the kHz range. Moreover, the invasive nature of most of these techniques hampers the study of cell mechanics evolution during biological processes under physiological conditions. Here, we report the use of laser-generated GHz acoustic waves to probe the stiffness and viscosity of nuclei in single live cells. We demonstrate that the stiffness and viscosity reflect the compressional dynamics of the nuclear components. Furthermore we reveal the existence of a universal nuclear stiffness equal to 15 GPa in adult mammalian cells. We also emphasize the importance of Poisson's ratio in the describing the dynamic mechanical behavior of cells. Accordingly, we postulate that at GHz acoustical frequencies, anharmonic processes might occur in the cell in addition to thermally activated absorption processes already considered at low frequencies. On this basis, we demonstrate that the mechanical properties of the internal structure of the cell nucleus probed by GHz acoustic waves correlate with a specific gene expression pattern during cell differentiation. We suggest that the induction of differentiation synchronizes the stiffness of the cell nuclei. This analysis is supported by the observation of a new stress fiber organization around the nucleus and of an increased number of focal adhesions throughout the entire cell. The method described here is therefore capable of probing in a non-invasive manner the nanomechanical behavior of single live cell nuclei. This approach should open new areas in the investigation of physiological processes under biological conditions.

Cells have a complex composition that yields an intricate rheological behavior, appealing for measurements over a wide frequency range. However the existing techniques cannot exceed the kHz range, and rely for most on injected or contacting functionalized microprobes. Here, we report on an innovative non-contact optoacoustic probing of the mechanical properties of single cells at GHz acoustic frequencies under physiological conditions.We culture cells on a biocompatible Ti6Al4V metal alloy. Low-energy femtosecond laser pulses are focused at the cell-Ti6Al4V interface to a sub-µm spot. The ensuing ultrafast thermal dilatation of the metal launches a high-frequency sound pulse in the cell. Acoustic propagation is measured remotely with an ultrafast laser probe through Brillouin light scattering. This all-optical non-invasive technique offers a broad frequency range, extending up to 1 THz, a sub-µm lateral resolution and a nanometer in-depth resolution.For illustration, we here concentrate on the cell nucleus. Using the laser-generated GHz acoustic waves, we probe the stiffness and viscosity of the nuclei of various cell types. We demonstrate for the first time that, at GHz frequencies, the nucleus stiffness tends to a unique value, much larger than that observed at kHz frequencies. We demonstrate that this universal stiffness reflects the compressional dynamics of the nucleus components.We also show that the evolution of the mechanical properties of the nucleus during cell differentiation is correlated with a specific gene expression pattern. In the frame of soft glass rheology, we project the GHz mechanical properties of the differentiated nuclei to the kHz range. Comparison with the kHz data obtained by alternative techniques suggests the existence of a new absorption process appearing at GHz frequencies.This innovative technique defines a new class of experiments to enlighten cell mechanics in physiological conditions at a subcell scale.

The picosecond ultrasonics technique is used to investigate the viscoelastic properties of nucleus of fixed single osteoblast progenitor cells adhering on a titanium alloy substrate. A two-color probing picosecond ultrasonics and a fluorescence visualization setups were developed and combined to allow to distinguish subcomponents inside the cell under investigation. It opens the way for quantitative measurements of the viscoelastic properties of single cells and of their sub-micrometer thickness. It is shown that a blue probe, λ = 400 nm, is preferable to a red probe, λ = 800 nm, to perform these measurements with fixed sub-micrometer bone cells. 26 GHz acoustic frequencies are detected in cells as thin as 135 nm. A 1D analytical model of the acoustic generation and of the optical detection is used to describe the experimental results. The nucleus longitudinal elastic moduli (13–16 GPa) and dynamic viscosities (13–30 cP) are measured at high frequencies (GHz) from a time-frequency analysis of the experimental data of fixed single cells.

Biological objects are exquisitely sensitive to temperature variations and their mechanical characterization is often a challenge when using the picosecond ultrasonics technique. To reduce the laser-induced temperature rise, we place single biological cells on a thin metal transducer and we focus the laser beam that generates the acoustic waves at frequencies ≤ 150 GHz on the rear side of the transducer. The acoustic waves propagate through the transducer and are partially transmitted to the cell to create the so-called Brillouin oscillations. The frequency of these oscillations provides a direct measurement of the sound velocity. The simultaneous measurement of the acoustic reflection coefficient at the transducer/cell interface allows the determination of both the density and the compressibility of the cell.

The measurement of the mechanical properties of single biological cells with a nanometer depth resolution using only coherent light is proposed. A pump-probe set-up based on an ultrafast laser (100 fs pulses) is used to excite and detect acoustic frequencies in the GHz range. Experiments are performed on single fixed mouse MC3T3 cells adhering on titanium alloy substrate. Using two different probe wavelengths, the contributions to the optical detection resulting from the cell interface displacements and from interactions between acoustic waves and the laser light are identified. Semi-analytical calculations allow the determination of acoustic celerities and thicknesses in cells thinner than 150 nm.

By controlling processing parameters such as the average fluence, number of laser pulses and beam polarization direction, different types of multiscale surface textures were produced on Ti-6Al-4V surfaces by ultrafast laser processing. The samples were textured in ambient atmosphere using an Yb: KYW chirped-pulse-regenerative amplification laser with a wavelength of 1030 nm and pulse duration of 500 fs. The wetting of simulated biological fluids as well as the human mesenchymal stem cells (hMSCs) behavior were assessed. Three types of textured surfaces were tested, consisting of: (i) Laser-Induced Periodic Surface Structures-LIPSS; (ii) nanopillars-like structures; and (iii) LIPSS overlapped to microcolumns. The laser textured surfaces present hydrophilic behavior and high affinity for HBSS (Hank’s balanced salt solution). Cell spreading and adhesion strength is reduced by the laser nanotextures as compared to a polished control surface. Cytoskeleton stretching and stress fibers were clearly observed on LIPSS while significant filopodia formation was verified on nanopillars. There was no cell proliferation on the laser nanotextured surfaces. Ultrafast laser texturing of Ti-6Al-4V surfaces is an efficient technique for increasing surface wettability, and is potentially useful as a technique to control the behavior of hMSCs by changing the cytoskeleton shape, FAPs distribution and area, and proliferation.By controlling processing parameters such as the average fluence, number of laser pulses and beam polarization direction, different types of multiscale surface textures were produced on Ti-6Al-4V surfaces by ultrafast laser processing. The samples were textured in ambient atmosphere using an Yb: KYW chirped-pulse-regenerative amplification laser with a wavelength of 1030 nm and pulse duration of 500 fs. The wetting of simulated biological fluids as well as the human mesenchymal stem cells (hMSCs) behavior were assessed. Three types of textured surfaces were tested, consisting of: (i) Laser-Induced Periodic Surface Structures-LIPSS; (ii) nanopillars-like structures; and (iii) LIPSS overlapped to microcolumns. The laser textured surfaces present hydrophilic behavior and high affinity for HBSS (Hank’s balanced salt solution). Cell spreading and adhesion strength is reduced by the laser nanotextures as compared to a polished control surface. Cytoskeleton stretching and stress fibers were clearly observed on L...