Contactless mechanical stimulation of the skin using shear waves.

The skin, the outermost organ of the human body, is vital for sensing and responding to stimuli through mechanotransduction. It is constantly exposed to mechanical stress. Consequently, various mechanical therapies, including compression, massage, and microneedling, have become routine practices for skin healing and regeneration. However, these traditional methods require direct skin contact, restricting their applicability. To address this constraint, we developed shear wave stimulation (SWS), a contactless mechanical stimulation technique. The effectiveness of SWS was compared with that of a commercial compression bioreactor used on reconstructed skin at various stages of maturity. Despite the distinct stimulus conditions applied by the two methods, SWS yielded remarkable outcomes, similar to the effects of the compression bioreactor. It significantly increased the shear modulus of tissue-engineered skin, heightened the density of collagen and elastin fibers, and resulted in an augmentation of fibroblasts in terms of their number and length. Notably, SWS exhibited diverse effects in the low- and high-frequency modes, highlighting the importance of fine-tuning the stimulus intensity. These results unequivocally demonstrated the capability of SWS to enhance the mechanical functions of the skin in vitro, making it a promising option for addressing wound healing and stretch mark recovery.

Key topographic parameters driving surface adhesion of Porphyromonas gingivalis.

Dental implant failure is primarily due to peri-implantitis, a consequence of bacterial biofilm formation. Bacterial adhesion is strongly linked to micro-/nano-topographies of a surface; thus an assessment of surface texture parameters is essential to understand bacterial adhesion. In this study, mirror polished titanium samples (Ti6Al4V) were irradiated with a femtosecond laser (fs-L) at a wavelength of 1030 nm (infrared) with variable laser parameters (laser beam polarization, number, spacing and organization of the impacts). Images of 3-D topographies were obtained by focal variation microscopy and analyzed with MountainsMap software to measure surface parameters. From bacteria associated with peri-implantitis, we selected Porphyromonas gingivalis to evaluate its adhesion on Ti6Al4V surfaces in an in vitro study. Correlations between various surface parameters and P. gingivalis adhesion were investigated. We discovered that Sa value, a common measure of surface roughness, was not sufficient in describing the complexity of these fs-L treated surfaces and their bacterial interaction. We found that Sku, density and mean depths of the furrows, were the most accurate parameters for this purpose. These results provide important information that could help anticipate the bacterial adhesive properties of a surface based on its topographic parameters, thus the development of promising laser designed biofunctional implants.

Polarization of Femtosecond Laser for Titanium Alloy Nanopatterning Influences Osteoblastic Differentiation.

Ultrashort pulse lasers have significant advantages over conventional continuous wave and long pulse lasers for the texturing of metallic surfaces, especially for nanoscale surface structure patterning. Furthermore, ultrafast laser beam polarization allows for the precise control of the spatial alignment of nanotextures imprinted on titanium-based implant surfaces. In this article, we report the biological effect of beam polarization on human mesenchymal stem cell differentiation. We created, on polished titanium-6aluminum-4vanadium (Ti-6Al-4V) plates, a laser-induced periodic surface structure (LIPSS) using linear or azimuthal polarization of infrared beams to generate linear or radial LIPSS, respectively. The main difference between the two surfaces was the microstructural anisotropy of the linear LIPSS and the isotropy of the radial LIPSS. At 7 d post seeding, cells on the radial LIPSS surface showed the highest extracellular fibronectin production. At 14 days, qRT-PCR showed on the same surface an increase in osteogenesis-related genes, such as alkaline phosphatase and osterix. At 21 d, mineralization clusters indicative of final osteoinduction were more abundant on the radial LIPSS. Taken together, we identified that creating more isotropic than linear surfaces enhances cell differentiation, resulting in an improved osseointegration. Thus, the fine tuning of ultrashort pulse lasers may be a promising new route for the functionalization of medical implants.

Extracellular matrix produced by osteoblasts cultured under low-magnitude, high-frequency stimulation is favourable to osteogenic differentiation of mesenchymal stem cells.

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 µÎµ), 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.

In vitro histomechanical effects of enzymatic degradation in carotid arteries during inflation tests with pulsatile loading.

In this paper, the objective is to assess the histomechanical effects of collagen proteolysis in arteries under loading conditions reproducing in vivo environment. Thirteen segments of common porcine carotid arteries (8 proximal and 5 distal) were immersed in a bath of bacterial collagenase and tested with a pulsatile tension/inflation machine. Diameter, pressure and axial load were monitored throughout the tests and used to derive the stress-stretch curves and to determine the secant circumferential stiffness. Results were analysed separately for proximal and distal segments, before and after 1, 2 and 3 h of enzymatic degradation. A histological analysis was performed to relate the arterial microstructure to its mechanical behavior under collagen proteolysis. Control (before enzymatic degradation) and treated populations (after 1, 2 or 3 h of enzymatic degradation) were found statistically incomparable, and histology confirmed the alteration of the fibrous structure of collagen bundles induced by the collagenase treatment. A decrease of the secant circumferential stiffness of the arterial wall was noticed mostly at the beginning of the treatment, and was less pronounced after 1 h. These results constitute an important set of enzymatically damaged arteries that can be used to validate biomechanical computational models correlating structure and properties of blood vessels.

Ultrafast Laser Processing of Nanostructured Patterns for the Control of Cell Adhesion and Migration on Titanium Alloy.

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.

Laser-Based Hybrid Manufacturing of Endosseous Implants: Optimized Titanium Surfaces for Enhancing Osteogenic Differentiation of Human Mesenchymal Stem Cells.

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 post-treatments.

Macrotopographic closure promotes tissue growth and osteogenesis in vitro.

While the impact of substrate topographies at nano- and microscale on bone cell behavior has been particularly well documented, very few studies have analyzed the role of substrate closure at a tissular level. Moreover, these have focused on matrix deposition rather than on osteoblastic differentiation. In the present work, mouse calvaria cells were grown for 15days on hydroxyapatite (HA) ceramics textured with three different macrogrooves shapes (**100µm): 1 sine and 2 triangle waveforms. We found that macrotopography favors cell attachment, and that bone-like tissue growth and organization are promoted by a tight "closure angle" of the substrate geometry. Interestingly, while Flat HA controls showed little marker expression at the end of the culture, cells grown on macrogrooves, and in particular the most closed (triangle waveform with a 517µm spatial period) showed a fast time-course of osteoblast differentiation, reaching high levels of gene and protein expression of osteocalcin and sclerostin, a marker of osteocytes. STATEMENT OF SIGNIFICANCE: Many in vitro studies have been conducted on topography at nano and microscale, fewer have focused on the influence of macrotopography on osteoblasts. Ceramics with a controlled architecture were obtained throught a 3D printing process and used to assess osteoblast behavior. Biocompatible, they allowed the long-terme survival of osteoblast cells and the laying of an important bone matrix. V-shaped grooves were found to accelerates osteoblast differentiation and promote bone-like tissue deposition and maturation (osteocyte formation), proportionately to angle closure. Such macrostructures are attractive for the design of innovative implants for bone tissue engineering and in vitro models of osteogenesis.

Comparison of four methods of surface roughness assessment of corneal stromal bed after lamellar cutting.

Corneal lamellar cutting with a blade or femtosecond laser (FSL) is commonly used during refractive surgery and corneal grafts. Surface roughness of the cutting plane influences postoperative visual acuity but is difficult to assess reliably. For the first time, we compared chromatic confocal microscopy (CCM) with scanning electron microscopy, atomic force microscopy (AFM) and focus-variation microscopy (FVM) to characterize surfaces of variable roughness after FSL cutting. The small area allowed by AFM hinders conclusive roughness analysis, especially with irregular cuts. FVM does not always differentiate between smooth and rough surfaces. Finally, CCM allows analysis of large surfaces and differentiates between surface states.

Femtosecond laser nano/micro patterning of titanium influences mesenchymal stem cell adhesion and commitment.

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.

Multiscale grooved titanium processed with femtosecond laser influences mesenchymal stem cell morphology, adhesion, and matrix organization.

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.

The effect of dual frequency cyclic compression on matrix deposition by osteoblast-like cells grown in 3D scaffolds and on modulation of VEGF variant expression.

As a strategy to optimise osteointegration of biomaterials by inducing proper extracellular matrix synthesis, and specifically angiogenic growth factor production and storage, we tested the effects of cyclic mechanical compression on 3D cultures of human osteoblast-like cells. MG-63 cells were seeded into 3D porous hydroxyapatite ceramics under vacuum to enable a homogenous cellular distribution. A four-day culture period allowed cell proliferation throughout the scaffolds. Low amplitude cyclic compressions were then applied to the scaffolds for 15 min with different regimens generated by the ZetOS system. A 3 Hz sinusoidal (sine) signal increased slightly collagen and fibronectin expression. When 50 Hz or 100 Hz vibrations were superimposed to the 3 Hz signal, matrix protein expression was down-regulated. In contrast, adding a 25 Hz vibration up-regulated significantly collagen and fibronectin. Moreover, expression of a matrix-bound variant of vascular endothelial growth factor-A (VEGF-A) was specifically stimulated compared to control or 3 Hz sine, and non-soluble VEGF protein was increased. Our study enabled us to identify low-amplitude, high-frequency strain regimen able to increase major matrix proteins of bone tissue and to regulate the expression of VEGF variants, showing that an appropriate combined loading has the potential to functionalise cellularized bone-like constructs.