A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium.

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

How biophysical in vivo testing techniques can be used to characterize full thickness skin equivalents.

BACKGROUND: The reliability of the biophysical properties of skin equivalents (SEs) remains a challenge for medical applications and for product efficacy tests following the European Directive 2003/15/EC2 on the prohibition of animal experiments for cosmetic products. METHODS: We propose to adapt the biophysical in vivo testing techniques to compare full thickness model growth vs. time. The interest in using such techniques lies in possible comparisons between in vivo and in vitro skin as well as monitoring samples over the culture time. RESULTS: High frequency ultrasound technique, optical coherence tomography (OCT), and laser scanning microscopy were used to analyze SEs morphology at days D42 and D60 whereas their microstructure was assessed through transmission electron microscopy and classical histology. A correlation between these observations and mechanical measurements has been proposed so as to underline the consequence of both the development of the dermis elastic fibers and the epidermis differentiation. CONCLUSION: Ultrasounds measurements show a highly homogeneous dermis whereas the OCT technique clearly distinguishes the stratum corneum and the living epidermis. The increase in the thicknesses of these layers as well as the growth in elastin and collagen fibers results in strong modifications of the samples mechanical properties.

A multitechnique evaluation of topical corticosteroid treatment.

BACKGROUND/PURPOSE: Corticosteroids are widely prescribed for systemic or local treatment of inflammatory autoimmune disorders. Long-term therapy is associated with side effects and causes cutaneous atrophy of the epidermis and the dermis. The present study aims to evaluate with several noninvasive techniques, the skin modifications observed during corticosteroids treatment. The potential of skin mechanical measurement and ultrasound radio frequency (RF) signal analysis are proposed as new measures more closely related to the functional impairments. METHODS: Thirteen young healthy women volunteers had two applications per day on one arm of topical Clobetasol propionate 0.05% for 28 days, and they were followed for 28 days more. Skin modifications were studied by high-frequency ultrasound imaging, ultrasound RF signal analysis, optical coherence tomography and by the suction test. RESULTS: For all the techniques, a statistically significant change is observed with treatment. Large variations, around 30%, are observed for all techniques, but less for ultrasound imaging (10%). Dermis and epidermis thickness presented stable measurements on the nontreated zone. At the end of the study, measures returned to normal. The dynamic is mainly observed within the first 14 days of treatment and within the first 14 days after its cessation. CONCLUSION: Similar dynamics of skin modification during corticosteroid treatment was observed with very different techniques. Moreover, the potential of RF ultrasound analysis and mechanical skin measurement for characterizing skin structural and functional impairments has been evaluated.

A nonlinear elastic behavior to identify the mechanical parameters of human skin in vivo.

BACKGROUND/PURPOSE: Various analyses have been performed to identify the mechanical properties of the human skin tissue in vivo. They generally use different approaches and hypotheses (behavior laws as well as mechanical tests) and the obtained results are consequently difficult to analyze and compare. In this paper, an inverse method that can be adapted to any kind of mechanical tests and behavior laws is presented. METHOD: A suction deformation performed on the volar aspect of the forearm of a subject is considered. This test is modeled with the finite element method to compare the experimental and simulated curves using an inverse method that allows the skin mechanical parameters identification. This process is based on two optimization algorithms, Kalman's filter and Gauss-Newton's methods. To account for the nonlinear behavior of the skin, a specific nonlinear elastic law, which is then compared with standard linear elastic and neo-Hookean's mechanical behaviors, was developed. RESULTS: The obtained results first prove that neither linear elasticity nor neo-Hookean's laws can be used to model the skin. On the contrary, the nonlinear elastic model presents a relevant fit of the experimental curves. The skin thickness is also proved to be another key point to be taken into consideration. CONCLUSIONS: The obtained results are successfully compared with literature and the reliability of the proposed method is underlined with the identification of 300 additional experimental curves. The different works we are currently focusing on are finally introduced.

Dual-parameter optimisation of the elastic properties of skin.

This paper presents a procedure for characterising the mechanical properties of skin using stochastic inverse identification. It is based on the minimisation of a cost function relative to the comparison between experimental suction experiments and their corresponding finite element models. Two different models are compared: a classical single-layer approach and a dual-layer medium which account for both the dermis and the hypodermis. Finite element results are used to construct the pre-optimisation database which is required for the inverse analysis. To compare the calculations, the entire identification is based on a dual-parameter optimisation procedure: for the single-layer approach a quadratic hyperelastic constitutive equation is used, whereas for the dual-layer medium a simple neo-Hookean potential is used. Theoretical conclusions, which are developed first, are then compared with actual case studies.