Gelatine-collagen photo-crosslinkable 3D matrixes for skin regeneration.

Immediate care of skin wounds and burns is essential to repair this mechanical and chemical barrier to infections. Hydrogels have become one of the standard methods for wound care. Here, gelatine-collagen photo-crosslinkable matrixes or hydrogels were manufactured by two-photon polymerization (TPP) or one-photon UV exposure using a Digital Light Processing (DLP) setup. Both techniques are able to construct matrixes from computer-aided design models, which is important for future clinical applications in which wound dressings should be customized. Although TPP can mimic the 3D dermo-epidermal junction with a high spatial resolution (i.e., ∼6 µm3), the manufacturing time was too slow to produce large wound dressings. Therefore, a DLP setup was explored in this study to fabricate large 2D matrixes of several cm2 using the same photo-resist as for TPP, except for the photoinitiator. The fibroblast viability, adherence, and proliferation were analysed in time on both 3D and 2D matrixes in vitro using two-photon microscopy. For both types of matrixes, the adherence and proliferation of fibroblasts (3T3-NIH) were optimal for stiff structures with a Young's modulus of 191 ± 35 kPa compared to softer matrixes of 37 ± 12 kPa. Fibroblast showed complete confluence on Day 14 after seeding on these matrixes, which may create the granulation tissue composed of fibronectin, collagen, and various proteoglycans in the future dermis before repair of the epidermis and disintegrating of their host matrix. For the monitoring of this repair, gelatine-collagen matrixes can easily incorporate bio-optical sensors for the simultaneous monitoring of inflammation processes and wound healing in time.

3D two-photon polymerization of smart cell gelatin - collagen matrixes with incorporated ruthenium complexes for the monitoring of local oxygen tensions.

The extra cellular matrix plays a major role in the biomechanical properties of tissues that impact cell behavior and fate. It is therefore crucial to mimic these complex cell-matrix interactions in 3D cell cultures. Here, two-photon polymerization is applied to produce gelatin methacryloyl (GelMA) - collagen matrixes that further enable local pO2matrix measurement, when ruthenium complexes are used as photo-activators. The fluorescence intensity of these complexes has a direct and inverse relationship with the local pO2matrix. The 3D structures reached their maximum size in cell culture conditions after 3H with a swelling factor of ~1.5. Their shape and the ruthenium fluorescence intensity of the alveoli walls stayed constant for at least 2 weeks in the absence of cells. They were used in time series to monitor the local pO2matrix adjacent to cancer cells during their division, migration and the formation of a tumor tissue mass. At the presence of these cell activities that consume O2, a significant ~3-fold increase of the ruthenium fluorescence intensity in the alveoli walls was observed. This study demonstrates that online monitoring of the local pO2matrix is possible. The ruthenium complexes provide the bio-optical sensors that are useful for further analysis of cancer and healthy cell energy metabolism in a 3D matrix that better mimics in vivo conditions and migration paths. Unraveling the cancer cell metabolic adaptations in a changing micro-environment will help the development of new therapeutic opportunities. STATEMENT OF SIGNIFICANCE: In 3D cell cultures, monitoring pericellular pO2 is as critical as controlling pH. This facility is currently missing. Here, we take advantage of the direct and inverse relationship between pO2 and the fluorescence intensity of ruthenium complexes to generate stable gelatin-collagen matrixes able to continuously monitoring the pO2 at the pericellular level. The ruthenium complexes, which are photo-activators in the two-photon polymerization of these matrixes, became covalently bind to the collagen fibers. Indeed, local O2 consumption by cancer cells during migration, mitosis and tumor mass formation caused a 3-fold increase of the ruthenium fluorescence. In the future, incorporating ruthenium complexes with other bio-optical sensors will create new drug screening platforms that monitor cell culture parameters at the pericellular level.

Polyhedral Bubble Vibrations.

Underwater bubbles are extremely good acoustic resonators, but are freely evolving and dissolving. Recently it was found that bubbles can be stabilized in frames, but the influence of the frame shape is still undocumented. Here we first explore the vibration of polyhedral bubbles with a low number of faces, shaped as the five Platonic solids. Their resonance frequency is well approximated by the formula for spherical bubbles with the same volume. Then we extend these results to shapes with a larger number of faces using fullerenes, paving the way to obtain arbitrary large resonant bubbles.

Acoustic interaction between 3D-fabricated cubic bubbles.

Spherical bubbles are notoriously difficult to hold in specific arrangements in water and tend to dissolve over time. Here, using stereolithographic printing, we built an assembly of millimetric cubic frames overcoming these limitations. Indeed, each of these open frames holds an air bubble when immersed into water, resulting in bubbles that are stable for a long time and are still able to oscillate acoustically. Several bubbles can be placed in any wanted spatial arrangement, thanks to the fabrication process. We show that bubbles are coupled acoustically when disposed along lines, planes or in 3D arrangements, and that their collective resonance frequency is shifted to much lower values, especially for 3D arrangements where bubbles have a higher number of close neighbours. Considering that these cubic bubbles behave acoustically as spherical bubbles of the same volume, we develop a theory allowing one to predict the acoustical emission of any arbitrary group of bubbles, in agreement with experimental results.

Multi-directional bubble generated streaming flows.

In previous work, we have demonstrated the use of single-holed Armoured Microbubbles (AMBs) for microfluidic mixing and self-propulsion. AMBs are hollow partial spheres, inside which we capture a bubble. Under ultrasound, the bubble oscillates, generating a streaming flow with velocities of 1-100 mm/s in water. In this paper, inspired by our successful fabrication of a C60 geometry (buckyball), we study AMBs with multiple surface holes. We show more holes generate additional pairs of fast circulations around the AMB. However, as the number of holes increases further, the circulations become small and the in-plane flow is dominated by a source or sink flow. For an AMB with two different sized holes, we demonstrate each hole can be independently activated, potentially useful for multi-directional swimming.

Acoustics of Cubic Bubbles: Six Coupled Oscillators.

We introduce cubic bubbles that are pinned to 3D printed millimetric frames immersed in water. Cubic bubbles are more stable over time and space than standard spherical bubbles, while still allowing large oscillations of their faces. We find that each face can be described as a harmonic oscillator coupled to the other ones. These resonators are coupled by the gas inside the cube but also by acoustic interactions in the liquid. We provide an analytical model and 3D numerical simulations predicting the resonance with very good agreement. Acoustically, cubic bubbles prove to be good monopole subwavelength emitters, with nonemissive secondary surface modes.

Bubble-based acoustic micropropulsors: active surfaces and mixers.

Acoustic micropropulsors present great potential for microfluidic applications. The propulsion is based on encapsulated 20 µm bubbles excited by a contacless ultrasonic transducer. The vibrating bubbles then generate a powerful streaming flow, with speeds 1-100 mm s-1 in water, through the action of viscous stresses. In this paper we introduce a full toolbox of micropropulsors using a versatile three-dimensional (3D) microfabrication setup. Doublets and triplets of propulsors are introduced, and the flows they generate are predicted by a theoretical hydrodynamic model. We then introduce whole surfaces covered with propulsors, which we term active surfaces. These surfaces are excited by a single ultrasonic wave, can generate collective flows and may be harnessed for mixing purposes. Several patterns of propulsors are tested, and the flows produced by the two most efficient mixers are predicted by a simple theoretical model based on flow singularities. In particular, the vortices generated by the most efficient pattern, an L-shaped mixer, are analysed in detail.

Obstructive micro diffracting structures as an alternative to plasmonics nano slits for making efficient microlenses.

Miniature optical components at the wavelength scale remain today a theoretically opened challenging problem of great technological interest. Appart from refractive micro-optics, plasmonics have been proposed to realize micro lenses with properly designed planar metallic nano-patterns. We show in this paper that efficient light focusing at the diffraction limit with higher transmission can be obtained with micro-structures much easier to fabricate than nano ones, such as a simple micro-slit studied here as an example. Optical properties are attributed to diffraction and a quantitative excellent agreement between experiment and theory is obtained.

Metallic nanowires can lead to wavelength-scale microlenses and microlens arrays.

We theoretically and experimentally demonstrate that the diffraction of microstructures based on silver nanowires leads to very efficient microfocusing effects. Pairs of parallel nanowires act as ultrasmall cylindrical microlenses with diffraction-limited resolution in the Fresnel region. This is a new diffraction scheme to make micron-sized optical lenses with higher transmittance than plasmonic microlens based on nano-aperture arrays. Calculations based on the scalar Rayleigh-Sommerfeld integral highlights the pure scalar diffractive contribution. Thus, the plasmon contribution is negligible in such micron-sized metallic geometry. We demonstrate that two-dimensional grids of nanowires can be used to fabricate dense arrays of microlenses, i.e. 10000x10000 DPI (dots per inch).

Tetranuclear manganese(II) complexes of sulfonylcalix[4]arene macrocycles: synthesis, structure, spectroscopic and magnetic properties.

Two tetranuclear manganese(II) complexes {K(+)[Mn(4)(ThiaSO(2))(2)(OH)](-)} (1) and {K(+)[Mn(4)(ThiaSO(2))(2)(F)](-)} (2) have been synthesized under solvothermal conditions in methanol with p-tert-butylsulfonylcalix[4]arene (ThiaSO(2)). For both complexes, the structure has been established from single-crystal X-ray diffraction. The two complexes are best described as manganese squares sandwiched between two thiacalixarene macrocycles. In both complexes, in the center of the square formed by the four manganese(II) atoms, the unexpected presence of µ(4)-OH(-) or µ(4)-F(-) gives a negative charge to the cluster. The two tetranuclear complexes exhibit strong orange luminescence behavior resulting from the symbiosis between the ThiaSO(2) and the Mn(2+). Despite similar chemical formulation, (1) and (2) present difference in emission intensity and lifetime τ.

Deep in vivo two-photon imaging of blood vessels with a new dye encapsulated in pluronic nanomicelles.

Our purpose is to test if Pluronic® fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.

Photodynamic therapy and two-photon bio-imaging applications of hydrophobic chromophores through amphiphilic polymer delivery.

The synthesis and photophysical properties of two lipophilic quadrupolar chromophores featuring anthracenyl (1) or dibromobenzene (2) were described. These two chromophores combined significant two-photon absorption cross-sections with high fluorescence quantum yield for 1 and improved singlet oxygen generation efficiency for 2, in organic solvents. The use of Pluronic nanoparticles allowed a simple and straightforward introduction of these lipophilic chromophores into biological cell media. Their internal distribution in various cell lines was studied using fluorescence microscopy and flow-cytometry following a successful staining that was achieved upon 2 h of incubation. Finally, multiphoton excitation microscopy and photodynamic therapy capability of the chromophores were demonstrated by cell exposure to a 820 nm fs laser and cell death upon one photon resonant irradiation at 436 ± 10 nm, respectively.

Electroless growth of silver nanoparticles into mesostructured silica block copolymer films.

Silver nanoparticles and silver nanowires have been grown inside mesostructured silica films obtained from block copolymers using two successive reduction steps: the first one involves a sodium borohydride reduction or a photoreduction of silver nitrate contained in the film, and the second one consists of a silver deposit on the primary nanoparticles, carried out by silver ion solution reduction with hydroxylamine chloride. We have demonstrated that the F127 block copolymer ((PEO)(106)(PPO)(70)(PEO)(106)), "F type", mesostructured silica film is a suitable "soft" template for the fabrication of spherical silver nanoparticles arrays. Silver spheres grow from 7 to 11 nm upon the second reduction step. As a consequence, a red shift of the surface plasmon resonance associated with metallic silver has been observed and attributed to plasmonic coupling between particles. Using a P123 block copolymer ((PEO)(20)(PPO)(70)(PEO)(20)), "P type", mesostructured silica film, we have obtained silver nanowires with typical dimension of 10 nm x 100 nm. The corresponding surface plasmon resonance is blue-shifted. The hydroxylamine chloride treatment appears to be efficient only when a previous chemical reduction is performed, assuming that the first sodium borohydride reduction induces a high concentration of silver nuclei in the first layer of the porous silica (film/air interface), which explains their reactivity for further growth.

Fluorescent Pluronic nanodots for in vivo two-photon imaging.

We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

Monodisperse fluorene oligomers exhibiting strong dipolar coupling interactions.

Well-defined fluorene oligomers (n = 1 to 6) were prepared step by step using Suzuki and Yamamoto couplings, while absorption and photoluminescence properties evidenced very large dipolar coupling interactions between fluorene moieties.