Skin and bone : the effect of age and estrogen deficiency.

Population ageing is a pervasive global phenomenon. For the elderly person, maintaining autonomy and independence are key to ensuring quality of life. Osteoporosis is a systemic skeletal disease characterised by low bone density andmicroarchitectural deterioration in bone tissue with a consequent increase in bonefragility. Epidemiological studies have highlighted the burden of disease on patient, society and health service. Dermatoporosis, previously considered a cosmetic and trivial issue, with increasing life expectancy, is a significant cause of morbidity. Theoverall goal of this thesis was to determine the effects of estrogen deficiency and age on bone and skin parameters, and to explore the potential of skin fragility as a surrogate to bone fragility in the diagnosis of osteoporosis. A successful ovine model of postmenopausal osteoporosis demonstrated significantly increased intracortical bone turnover, albeit in a seasonal manner, in response to estrogen deficiency. This was associated with a compensatory geometrical /structural adaptation of the distribution of bone mass. Estrogen deficiency is associated with a significantly increased intracortical porosity, however there is no adverse effect onbone compression strength at 31 months post ovariectomy. Zoledronic acid decreases bone turnover below that of controls and minimises geometrical /structural adaptation in response to estrogen deficiency. Geometrical analysis in a human cadaveric cohort demonstrated the effects of age and estrogen deficiency on the adaptive mechanisms in bone, with a concomitant decline in form and function of skin and bone with age. Qualitatively our data indicate that at a tissue level there is minimal difference between the bone of male and female,however the material properties of skin in male and female are significantly different. Geometrical analysis of the alterations in the periosteal and endosteal surfaces of cortical bone provide us with valuable information regarding the response and adaptation to sex steroid deficiency and age, however they alone do not give usgreater insight into fracture prediction. The relationship between BMD and BR demonstrates that despite the limitations of conventional BMD measurements, they do reflect, to an extent, the geometrical adaptation over time. Our study suggests that acritical balance between BMC and BR exists. Longitudinal analysis of BMD and geometrical changes, specifically buckling ratio and cortical thinning, will in the future provide for stratification of those at risk based on our understanding of howbone mass is redistributed over time. Raman spectroscopic analysis of bone and skin in an aged human cohort and ovine model of osteoporosis demonstrated common molecular changes in skin and bone. In vivo Raman spectroscopy is a technique likely to develop into a powerful method for future applications. In order to employ the small but significant differencesbetween normal and pathological samples as a diagnostic tool, the present results warrant further investigation and confirmation, to ultimately compile a data bank containing normal and abnormal spectra, combined with appropriate diagnostic algorithms for acceptance into medical practice

Raman Spectroscopic Analysis of Human Skin Tissue Sections Ex-vivo: Evaluation of the Effects of Tissue Processing and Dewaxing

Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections, and the effects of tissue processing. Both hand and thigh sections of human cadavers were analysed in their unprocessed and formalin fixed paraffin processed (FFPP) and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum, intermediate underlying epithelium and dermal layers for sections from both anatomical sites. The stratum corneum is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the stratum corneum, intermediate and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks

Analysis of Human Skin Tissue by Raman Microspectroscopy: Dealing with the Background

Raman microspectroscopy is widely used for molecular characterisation of tissue samples. Nevertheless, when working in vitro on tissue sections, the presence of a broad background to the spectra remains problematic and its removal requires advanced methods for pre-processing of the data. To date, research efforts have been primarily devoted to development of techniques of statistical analysis to extract the relevant information contained in the spectra. However, few attempts have been made to understand the origin of the background and to improve the protocols used for the collection of Raman spectra that could lead to the reduction or elimination of the background. It has been demonstrated that measurement at 785nm in water immersion significantly reduces the Raman background of both pure biochemical components and tissue sections, associating the background at 785nm with a scattering phenomenon rather than fluorescence. It is however of interest to probe the dependence of the observed background and any time evolution normally associated with photobleaching of fluorophores, under dry and immersed conditions, on the source wavelength. Using 785nm or 660nm as source, extended exposure of dried skin tissue sections to the laser results in a time dependent reduction of the background present in the Raman spectra. When working in water immersion, the overall background as well as the evolution over time is greatly reduced and the background is seen to stabilise after ~20 seconds exposure. Using 532 nm or 473 nm as source for the examination of dried tissue sections, visible photodamage of the sample limits the laser power usable for the collection of spectra to 5 mW. Immersion of the tissue sections protects against photodamage and laser powers of up to 30 mW can be used without any visible damage. Under these conditions, the background is significantly reduced and good quality Raman spectra can be recorded. By adapting the protocol usually used for the collection of Raman spectra, this study clearly demonstrates that other approaches rather than mathematical manipulation of the data can be used to deal with the intrinsic background commonly observable. Notably, the dependence of the background and its time evolution under prolonged exposure on sample environment potentially sheds light on its origin as due to sample morphology (scattering) rather than chemical content (fluorescence). Overall, the study demonstrates that, in addition to reduced background, the photostability of the samples is significantly enhanced in an immersion geometry

Autologous Microvascular Breast Reconstruction

Autologous microvascular breast reconstruction is widely accepted as a key component of breast cancer treatment. There are two basic donor sites; the anterior abdominal wall and the thigh/buttock region. Each of these regions provides for a number of flaps that are successfully utilised in breast reconstruction. Refinement of surgical technique and the drive towards minimising donor site morbidity whilst maximising flap vascularity in breast reconstruction has seen an evolution towards perforator based flap reconstructions, however myocutaneous flaps are still commonly practiced. We review herein the current methods of autologous microvascular breast reconstruction

Structural adaptation and intracortical bone turnover in an ovine model of osteoporosis.

Compact bone makes up approximately 80% of the human skeletal mass. This study examines the effect of estrogen deficiency on compact bone turnover and associated geometrical structural adaptation over a 31-month period in a large animal model. Twenty-seven skeletally mature sheep were divided into control (n = 16) and ovariectomy group (OVX, n = 11). Animals were administered five different fluorochrome dyes to label intracortical bone turnover, and sacrificed at 31 months. Compact bone samples were analyzed for cortical geometry, intracortical turnover at five time points, resorption cavities, porosity, and compressive strength. Intracortical bone turnover was significantly increased in OVX, which demonstrated seasonal variation. Cross-sectional area in OVX was significantly greater than control and was associated with an increased section modulus. Intracortical porosity was significantly increased in OVX, however, there was no significant difference in ultimate compressive strength between the groups. Our results demonstrate increased intracortical bone turnover, resportion spaces, and porosity in OVX, without adversely affecting compressive strength. Our results also support the hypothesis of geometrical adaptation of compact bone in response to estrogen deficiency. These results suggest an early structural compensatory response in compact bone, despite increased intracortical turnover

Raman spectroscopic analysis of human skin tissue sections ex-vivo : evaluation of the effects of tissue processing and dewaxing

International audienceRaman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections and the effects of tissue processing. Both hand and thigh sections of human cadavers were analyzed in their unprocessed and formalin-fixed, paraffin-processed (FFPP), and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum (SC), intermediate underlying epithelium, and dermal layers for sections from both anatomical sites. The SC is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the SC, intermediate, and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks