Simultaneous isolation of keratinocytes and fibroblasts from a human cutaneous biopsy for the production of autologous reconstructed skin

The key step In the reconstruction of skin by the self-assembly approach is to use fibroblasts capable of secreting a mature extracellular matrix and keratinocytes that can associate with one another to form a stratified, differentiated epidermis. To determine the most efficient way to extract both cell types from a single cutaneous biopsy, five different enzymatic combinations were tested. The yield, viability and morphology of the recuperated cells were compared. The length and temperature of the enzymatic treatments influenced the number and the type of cells recuperated. The cells were then cultured in appropriate media in monolayers and reconstructed skin were produced using the self-assembly approach. No difference was observed between the different strains of cells.La réussite de la technique d'auto-assemblage pour la reconstruction de la peau dépend de la capacité des fibroblastes à sécréter une matrice extracellulaire complexe ainsi que celle des kératinocytes à s'associer pour former un épiderme stratifié et différencié. Pour déterminer la manière la plus efficace d'extraire ces deux types cellulaires d'une seule biopsie cutané, cinq combinaisons d'enzymes ont été comparées. La durée et la température des traitements enzymatiques ont influencé le nombre de cellule récupérés. Les cellules ont d'abord été cultivées en monouche, puis peaux reconstruites par la méthode d'auto-assemblage ont été produites. Aucune différence n'a été observée entre les différentes Iignées

Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries

The survival of thick tissues/organs produced by tissue engineering requires rapid revascularization after grafting. Although capillary-like structures have been reconstituted in some engineered tissues, little is known about the interaction between normal epithelial cells and endothelial cells involved in the in vitro angiogenic process. In the present study, we used the self-assembly approach of tissue engineering to examine this relationship. An endothelialized tissue-engineered dermal substitute was produced by adding endothelial cells to the tissue-engineered dermal substitute produced by the self-assembly approach. The latter consists in culturing fibroblasts in the medium supplemented with serum and ascorbic acid. A network of tissue-engineered capillaries (TECs) formed within the human extracellular matrix produced by dermal fibroblasts. To determine whether epithelial cells modify TECs, the size and form of TECs were studied in the endothelialized tissue-engineered dermal substitute cultured in the presence or absence of epithelial cells. In the presence of normal keratinocytes from skin, cornea or uterine cervix, endothelial cells formed small TECs (cross-sectional area estimated at less than 50 μm2) reminiscent of capillaries found in the skin's microcirculation. In contrast, TECs grown in the absence of epithelial cells presented variable sizes (larger than 50 μm2), but the addition of keratinocyte-conditioned media or exogenous vascular endothelial growth factor induced their normalization toward a smaller size. Vascular endothelial growth factor neutralization inhibited the effect of keratinocyte-conditioned media. These results provide new direct evidence that normal human epithelial cells play a role in the regulation of the underlying TEC network, and advance our knowledge in tissue engineering for the production of TEC networks in vitro

Prospective study on the treatment of lower-extremity chronic venous and mixed ulcers using tissue-engineered skin substitute made by the self-assembly approach

BACKGROUND: Despite present optimal standard treatment of lower-extremity ulceration, a high incidence of recurrence and treatment failure is observed. The objective of this project was to evaluate the effect of a self-assembled skin substitute (SASS) made by tissue engineering as a temporary cutaneous dressing in the treatment of hard-to-heal chronic ulcers. PATIENTS AND METHODS: The prospective uncontrolled case study includes patients suffering from venous or mixed ulcers lasting more than 6 months and unresponsive to compression therapy, with an Ankle Brachial Index greater than 0.5. Compression therapy was combined with the weekly application of SASS, produced from the patient’s own skin cells, until healing. A weekly follow-up recorded wound size, skin aspect, pain, drainage, and percentage of wound healing. Photographs were also taken to assess ulcer evolution. RESULTS: Fourteen ulcers present on 5 patients were treated. A mean of 6.7 SASS depositions by ulcer was required for healing. Two ulcers developed a minor wound infection, which was treated with oral antibiotics; another 2 ulcers recurred, and 1 healed with a second course of treatment, whereas 1 ulcer had a small recurrence treated with local wound care. CONCLUSION: The authors’ study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy

Graduated Compression Stocking Thromboprophylaxis for Elderly Inpatients A Propensity Analysis

International audienceBACKGROUND: Graduated compression stockings (GCS) are often used for deep vein thrombosis prophylaxis in nonsurgical patients, although evidence on their effectiveness is lacking in this setting. OBJECTIVE: To determine whether prophylaxis with GCS is associated with a decrease in the rate of deep vein thrombosis in nonsurgical elderly patients. METHODS: Using original data from 2 multicenter nonrandomized studies, we performed multivariable and propensity score analyses to determine whether prophylaxis with GCS reduced the rate of deep vein thrombosis among 1,310 postacute care patients 65 years or older. The primary outcome was proximal deep vein thrombosis detected by routine compression ultrasonography performed by registered vascular physicians. RESULTS: Proximal deep vein thrombosis was found in 5.7% (21/371) of the GCS users and in 5.2% (49/939) of the GCS nonusers (odds ratio [OR], 1.09; 95% confidence interval [CI], 0.64-1.84). Although adjusting for propensity score eliminated all differences in baseline characteristics between users and nonusers, the OR for proximal deep vein thrombosis associated with GCS remained nonsignificant in propensity-stratified (adjusted OR, 1.11; 95% CI, 0.59-2.10) and propensity-matched (conditional OR, 0.92; 95% CI, 0.42-2.02) analysis. Similar figures were observed for distal and any deep vein thrombosis. The rates of deep vein thrombosis did not differ according to the length of stockings. CONCLUSIONS: Prophylaxis with GCS is not associated with a lower rate of deep vein thrombosis in nonsurgical elderly patients in routine practice. Randomized studies are needed to assess the efficacy of GCS when properly used in this setting

The MALINA oceanographic expedition: how do changesin ice cover, permafrost and UV radiation impactbiodiversity and biogeochemical fluxesin the Arctic Ocean?

International audienceThe MALINA oceanographic campaign was conducted during summer 2009 to investigate the carbon stocks and the processes controlling the carbon fluxes in the Mackenzie River estuary and the Beaufort Sea. Dur- ing the campaign, an extensive suite of physical, chemical and biological variables was measured across seven shelf–basin transects (south-north) to capture the meridional gradient between the estuary and the open ocean.Key variables such as temperature, absolute salinity, radiance, irradiance, nutrient concentrations, chlorophyll-a concentration, bacteria, phytoplankton and zooplankton abundance and taxonomy, and carbon stocks and fluxes were routinely measured onboard the Canadian research icebreaker CCGS Amundsen and from a barge in shallow coastal areas or for sampling within broken ice fields. Here, we present the results of a joint effort to tidy and standardize the collected data sets that will facilitate their reuse in further studies of the changing Arctic Ocean