Frédéric Cuisinier

Porous silicon scaffolds for stem cells growth and osteodifferentiation

Abstract: Stem cells constitute the source of differentiated cells for the generation of tissues during development and for regeneration of tissues that are diseased or injured postnatally. Stem cell-based therapy often requires a scaffold to carry cells and/or growth factor to the injured site. Porous silicon (PSi) is a promising biomaterial for tissue engineering, as it is both non-toxic and bioresorbable. Surface modification can offer control over the degradation rate of PSi and can also impart properties to promote cell adhesion. Thus, coupling the good proliferation and differentiation capacities of either adult mesenchymal stem cells (MSC) or embryonic stem cells (ESC) with the textural and chemical properties of the PSi substrates provides an interesting approach for therapeutic use. Moreover, the PSi nanostructure and the release of silicic acid have positive effect on precursor cell osteodifferentiation and mineralized matrix formation.

Raman confocal microscopy and AFM combined studies of cancerous cells treated with Paclitaxel

Paclitaxel interferes with the normal function of microtubule breakdown, induces apoptosis in cancer cells and sequesters free tubulin. As this drug acts also on other cell mechanisms it is important to monitor its accumulation in the cell compartments. The intracellular spreading of the drug was followed using a WITEC 300R confocal Raman microscope equipped with a CCD camera. Hence Atomic force microscopy (an MFP3D- Asylum Research AFM) in imaging and force mode was used to determine the morphological and mechanical modifications induced on living cells. These studies were performed on living epithelial MCF-7 breast cancer cells. Paclitaxel was added to cell culture media for 3, 6 and 9 hours. Among the specific paclitaxel Raman bands we selected the one at 1670 cm-1 because it is not superposed by the spectrum of the cells. Confocal Raman images are formed by monitoring this band, the NH2 and the PO4 band. Paclitaxel slightly accumulates in the nucleus forming patches. The drug is also concentrated in the vicinity of the cell membrane and in an area close to the nucleus where proteins accumulate. Our AFM images reveal that the treated cancerous MCF-7 cells keep the same size as the non treated ones, but their shape becomes more oval. Cells elasticity is also modified: a difference of 2 kPa in the Young Modulus characterizes the treated MCF-7 mammary cancerous cell. Our observations demonstrate that paclitaxel acts not only on microtubules but accumulates also in other cell compartments (nucleus) where microtubules are absent.

An 8-week, randomized, controlled, clinical study of the use of a 0.1% chlorhexidine mouthwash by chronic periodontitis patients.

AIM   To evaluate the efficacy of a 2-week administration of a 0.1% chlorhexidine mouthwash in the short-term treatment of chronic periodontitis patients and the impact of this product when administered twice by pocket irrigation. METHODS   Sixty patients were enrolled in a single-centre, placebo-controlled, randomized study with the blind allocation of product to two parallel groups. Clinical assessments were performed, and samples from six selected subgingival sites were collected for microbial analysis by culture at baseline, D15 and D56. Three of the six sites were randomly selected and were treated by subgingival irrigation with the same 0.1% chlorhexidine product at D0 and D7. A subsequent statistical analysis was performed using the paired Students t-test and Wilcoxon rank sum test for within-group analyses; analysis of variance and the Kruskall-Wallis test were used for between-group analyses. RESULTS   Two-week treatment with a 0.1% chlorhexidine mouthwash slightly reduced the gingival inflammation associated with periodontitis. We observed a significant decrease in Gram-negative, facultative anaerobes and micro-aerophiles, and a significant increase in Gram-positive cocci. No increase in the treatment effect was demonstrated by irrigation of the periodontal pockets. CONCLUSION   The 0.1% chlorhexidine mouthwash showed limited beneficial effects in the treatment of periodontitis patients.

A multitechnique study of bacteriorhodopsin's photonics toward new optical devices

Bacteriorhodopsin (BR) is a robust trans-membrane protein that functions as a light-driven proton pump, thus is an excellent candidate for biophotonics applications. For the development of new optical devices, the buildup of stable BR matrices has to be optimised. In this work, we present a multi- technique approach: the combination of optical waveguide lightmode spectroscopy (OWLS), atomic force microscopy (AFM) and multi-photon microscopy (MPM) aiming to analyze the optical and physico-chemical properties of BR embedded in polyelectrolyte multilayers (PEM) in its membrane bound form (purple membrane, PM), as well as solubilized BR immobilized within a photonic structure built of porous silicon (PSi). OWLS measurements revealed the possibility of incorporation of PM-BR layers into PE-multilayers. The calculated thickness and refractive index of the adsorbed layers demonstrate the successful adsorption of PM on top of the positively or negatively charged PE layers. Morphological studies by AFM proved a complete coverage of the positively charged PE layer with PM patches. As for the other model system, photonic responses of BR, after being immobilized within PSi substrates, have been evaluated using multi-photon microscopy. Fluorescence emission and second harmonic generation (SHG) of the BR-PSi system were observed at some particular pores of PSi and subsequent enhancement of the signal arising from the BR adsorbed within the pores was detected. Our results constitute the first steps of two interesting and innovative biomimetic approaches for the future design and development of BR based integrated optical devices.

Dental pulp stem cells (DPSCs) differentiation study by confocal Raman microscopy

Regenerative medicine brings a huge application for Mesenchymal stem cells such as Dental Pulp Stem Cells (DPSCs). Confocal Raman microscopy, a non-invasive, label free , real time and high spatial resolution imaging technique is used to study osteogenic differentiation of DPSCs. Integrated Raman intensities in the 2800-3000 cm-1 region (C-H stretching) and 960 cm-1 peak (phosphate PO4 3-) were collected. In Dental Pulp Stem Cells 21st day differentiated in buffer solution, phosphate peaks ν1 PO4 3- (first vibrational mode) at 960cm-1 and ν2 PO4 3- at 430cm–1 and ν4 PO4 3- at 585cm-1 are obviously present. Confocal Raman microscopy enables the detection of cell differentiation and it can be used to investigate clinical stem cell research.

Molecular detection via hybrid peptide-semiconductor photonic devices

The aim of this work was to investigate the possibilities to support device functionality that includes strongly confined and localized light emission and detection processes within nano/micro-structured semiconductors for biosensing applications. The interface between biological molecules and semiconductor surfaces, yet still under-explored is a key issue for improving biomolecular recognition in devices. We report on the use of adhesion peptides, elaborated via combinatorial phage-display libraries for controlled placement of biomolecules, leading to user-tailored hybrid photonic systems for molecular detection. An M13 bacteriophage library has been used to screen 1010 different peptides against various semiconductors to finally isolate specific peptides presenting a high binding capacity for the target surfaces. When used to functionalize porous silicon microcavities (PSiM) and GaAs/AlGaAs photonic crystals, we observe the formation of extremely thin (<1nm) peptide layers, hereby preserving the nanostructuration of the crystals. This is important to assure the photonic response of these tiny structures when they are functionalized by a biotinylated peptide layer and then used to capture streptavidin. Molecular detection was monitored via both linear and nonlinear optical measurements. Our linear reflectance spectra demonstrate an enhanced detection resolution via PSiM devices, when functionalized with the Si-specific peptide. Molecular capture at even lower concentrations (femtomols) is possible via the second harmonic generation of GaAs/AlGaAs photonic crystals when functionalized with GaAs-specific peptides. Our work demonstrates the outstanding value of adhesion peptides as interface linkers between semiconductors and biological molecules. They assure an enhanced molecular detection via both linear and nonlinear answers of photonic crystals.