No abstract

The femtosecond laser processing enabled the structuring of six types of surfaces on titanium-6aluminium-4vanadium (Ti-6Al-4V) plates. The obtained hierarchical features consisted of a combination of microgrooves and oriented nanostructures. By adjusting beam properties such as laser polarization, the width of the microgrooves (20 or 60 μm) and the orientation of the nanostructures (parallel or perpendicular to the microgrooves) can be precisely controlled. Mesenchymal stem cells (MSCs) grown on these structured surfaces produced cytoplasmic extensions with focal contacts, while on the smooth titanium, the cells were found to be well spread and without any focal contact 12 h postseeding. The 600-nm wide nanostructures on their own were sufficient to orient the MSCs. For the multiscale structured areas, when the orientation of the nanostructures was orthogonal in relation to the microgrooves, there was an important decrease in or even a loss of cell alignment signifying that cells were sensitive to the directional nanostructures in the microgrooves. At 7 days, cell proliferation was not affected but the direction of nanostructures controlled the matrix organization. The ultrafast laser, as a new method for producing micro-nanohybrid surfaces, is a promising approach to promote desired tissue organization for tissue engineering.

No abstract

Surface improvement of implants is essential for achieving a fast osseo-integration. Technically, the creation of a precise pattern on a titanium alloy surface is challenging. Here, the femtosecond laser was chosen as an innovative technology for texturing with accuracy a nano-micro topography. By adjusting the laser parameters, three biomimetic textures were fabricated on the titanium surface: micropits with nano-ripples in the pits, micropits with nano-ripples around the pits, and a texture with only nano-ripples. Mesenchymal stem cells (MSCs, C3H10T1/2) grown on these surfaces displayed altered morphometric parameters, and modified their focal adhesions in term of number, size, and distribution depending on surface type. These results indicate that the MSCs perceived subtle differences in topography. Dynamic analyses of early cellular events showed a higher speed of spreading on all the textured surfaces as opposed to the polished titanium. Concerning commitment, all the laser-treated surfaces strongly inhibited the expression of adipogenic-related genes (PPARϒ2, C/EBPα) and up-regulated the expression of osteoblastic-related genes (RUNX2, osteocalcin). Interestingly, the combination of micropits to nano-ripples enhanced their osteogenic potential as seen by a twofold increase in osteocalcin mRNA. Alkaline phosphatase activity was increased on all the textured surfaces, and lipid production was down-regulated. The functionalization of metallic surfaces by this high-resolution process will help us understand the MSCs' interactions with substrates for the development of textured implants with predictable tissue integrative properties.

The effects of low-magnitude, high-frequency (LMHF) mechanical stimulation on osteoblastic cells are poorly understood. We have developed a system that generates very small (15-40 με), high-frequency (400 Hz, sine) deformations on osteoblast cultures (MC3T3-E1). We investigated the effects of these LMHF stimulations mainly on extracellular matrix (ECM) synthesis. The functional properties of this ECM after decellularization were evaluated on C3H10T1/2 mesenchymal stem cells (MSCs). LMHF stimulations were applied 20 min once daily for 1, 3, or 7 days in MC3T3-E1 culture (1, 3, or 7 dLMHF). Cell number and viability were not affected after 3 or 7 dLMHF. Osteoblast response to LMHF was assessed by an increase in nitric oxide secretion, alteration of the cytoskeleton, and focal contacts. mRNA expression for fibronectin, osteopontin, bone sialoprotein, and type I collagen in LMHF cultures were 1.8-, 1.6-, 1.5-, and 1.7-fold higher than controls, respectively (P < 0.05). In terms of protein, osteopontin levels were increased after 3 dLMHF and ECM organization was altered as shown by fibronectin topology after 7 dLMHF. After decellularization, 7 dLMHF-ECM or control ECM was reseeded with MSCs. Seven dLMHF-ECM improved early events such as cell attachment (2 h) and focal contact adhesion (6 h) and, later (16 h), modified MSC morphological parameters. After 5 days in multipotential medium, gene-expression changes indicated that 7 dLMHF-ECM promoted the expression of osteoblast markers at the expense of adipogenic marker. LMHF stimulations of osteoblasts are therefore efficient and sufficient to generate osteogenic matrix.

No abstract

Femtosecond laser texturing is a promising surface functionalization technology to improve the integration and durability of dental and orthopedic implants. Four different surface topographies were obtained on titanium-6aluminum-4vanadium plates by varying laser processing parameters and strategies: surfaces presenting nanostructures such as laser-induced periodic surface structures (LIPSS) and ‘spikes’, associated or not with more complex multiscale geometries combining micro-pits, nanostructures and stretches of polished areas. After sterilization by heat treatment, LIPSS and spikes were characterized to be highly hydrophobic, whereas the original polished surfaces remained hydrophilic. Human mesenchymal stem cells (hMSCs) grown on simple nanostructured surfaces were found to spread less with an increased motility (velocity, acceleration, tortuosity), while on the complex surfaces, hMSCs decreased their migration when approaching the micro-pits and preferentially positioned their nucleus inside them. Moreover, focal adhesions of hMSCs were notably located on polished zones rather than on neighboring nanostructured areas where the protein adsorption was lower. All these observations indicated that hMSCs were spatially controlled and mechanically strained by the laser-induced topographies. The nanoscale structures influence surface wettability and protein adsorption and thus influence focal adhesions formation and finally induce shape-based mechanical constraints on cells, known to promote osteogenic differentiation.

Additive manufacturing (AM) is becoming increasingly important in the orthopedic and dental sectors thanks to two major advantages: the possibility of custom manufacturing and the integration of complex structures. However, at smaller scales, surface conditions of AM products are not mastered. Numerous non-fused powder particles give rise to roughness values (Sa) greater than 10 μm, thus limiting biomedical applications since the surface roughness of, e.g., metal implants plays a major role in the quality and rate of osseointegration. In this study, an innovative hybrid machine combining AM and a femtosecond laser (FS) was used to obtain Ti6Al4V parts with biofunctional surfaces. During the manufacturing process, the FS laser beam "neatly" ablates the surface, leaving in its path nanostructures created by the laser/ matter interaction. This step decreases the Sa from 11 to 4 μm and increases the surface wettability. The behavior of human mesenchymal stem cells was evaluated on these new AM+FS surfaces and compared with that on AM surfaces and also on polished surfaces. The number of cells attached 24 h after plating is equivalent on all surfaces, but cell spreading is higher on AM+FS surfaces compared with their AM counterparts. In the longer term (days 7 and 14), fibronectin and collagen synthesis increase on AM+FS surfaces as opposed to AM alone. Alkaline phosphatase activity, osteocalcin production, and mineralization, markers of osteogenic differentiation, are significantly lower on raw AM surfaces, whereas on the AM+FS specimens they display a level equivalent to that on the polished surface. Overall, these results indicate that using an FS laser beam during the fabrication of AM parts optimizes surface morphology to favor osteoblastic differentiation. This new hybrid machine could make it possible to produce AM implants with functional surfaces directly at the end of AM, thereby limiting their posttreatments.