Prolonged viability of human organotypic skin explant in culture method (hOSEC)

Abstract BACKGROUND: Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests. OBJECTIVES: This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes' proliferative capacity up to 75 days in culture. METHODS: The skin explants obtained from surgeries were cultured in CO2-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day. RESULTS: On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67. CONCLUSION: The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses. .

A comparison between sphygmomanometer-based and ambulatory blood pressure monitoring in acute salt loading and depletion protocol

INTRODUCTION: Ambulatory blood pressure monitors have been used in salt loading and depletion protocols. However, the agreement between measurements made using ambulatory blood pressure monitors and those made with the sphygmomanometer has not been evaluated. OBJECTIVE: The objective of this study was to compare the concordance of the two methods of blood pressure measurements in protocols of acute salt loading and depletion. METHOD: Systolic blood pressure was measured using a sphygmomanometer at the completion of salt infusion (2 L NaCl 0.9 percent, 4 h) and salt depletion (furosemide, 120mg/day, p.o.) in 18 volunteers. Using the Pearson correlation coefficient (ρ), these readings were compared with the mean systolic blood pressure measured using the ambulatory blood pressure monitoring device during the following periods: 4 h of saline infusion and 12 h of salt depletion; 4 h of saline infusion and the last 6 h of salt depletion; 12 h of salt loading and the last 6 h of depletion; 12 h of salt loading and 12 h of depletion. Salt sensitivity was defined by a difference in the systolic blood pressure between salt loading and salt depletion greater than 10 mmHg when measured with the sphygmomanometer, and the Kappa analysis of concordance (K) was used with a significance level of P<0.05. RESULTS: Only the blood pressure readings obtained using the ambulatory blood pressure device during 4 h of intravenous NaCl and during 12 h of salt depletion showed a high correlation with the variation in the systolic blood pressure measured by the sphygmomanometer, with a full agreement with the salt sensitivity classification (p = 0.71; P = 0.001 and K=1). CONCLUSION: In acute salt loading and depletion protocols, an ambulatory blood pressure monitoring device should be used to record the blood pressure during the 4-h interval of salt infusion and 12-h interval of salt depletion.