RESUMO
Objectives: The aim of this study was to evaluate cellular indicators, which change with exposure to ultraviolet (UV) radiation and can be used as parameters for measuring sunscreens efficiency. Methods: Commercial strains of L929 and HaCaT cells (skin dermis and epidermis, respectively), from the cell bank of Rio de Janeiro, were exposed to different doses of UVA (350 nm) and UVB (309 nm) radiation. The evaluation of the photoprotective potential of sunscreens was analyzed with cell viability, lipid peroxidation and ROS generation tests. Samples of sunscreen with SPF values ranging from 15 to 60 were applied to a quartz plate superimposed on the top of a microplate containing the cell culture, and then the system was irradiated. Results: The viability and lipid peroxidation of the two cell lines remained unchanged after exposure to UVA radiation. When exposed to UVB radiation, the reduction in viability and the increase in lipid peroxides were dose-dependent, that is, they varied from 3.15% to 95.4%, and from 1.2 to 42.7 nM MDA/pg protein, respectively, both for the L929 strain. The dose of 0.5 J/cm2 reduced by 41.4%±1.67 the number of viable cells, and the dose of 30 J/cm2 promoted the oxidation of 42.7 nM of MDA/pg protein. These doses were selected to evaluate the photoprotective effectiveness of commercial sunscreens. Sunscreens exposed to UVB rays could prevent the loss of cell viability (viability remained around 100% for higher SPF) and the formation of lipid peroxides (30 to 80% reduction of peroxide levels). None of the two cell strains, submitted to UVB radiation, formed amounts of intracellular ROS in a dose-dependent manner. Under exposure to UVA radiation, only the HaCaT cell line produced the largest amounts of ROS in a dose-dependent manner. After treating these cells with photoprotective formulations (20 J/cm2), the researchers observed a reduction in the amount of ROS formed. Conclusions: The parameters of cell viability and lipid peroxidation were promising to evaluate the photoprotective capacity of sunscreens against UVB radiation. The generation of ROS expressed in the HaCaT strain can discriminate the photoprotective potential of formulations against UVA radiation, as sunscreens reduced the formation of ROS. These results suggest that in vitro tests that evaluate the damage caused to cells can predict cellular indicators of the photoprotective effectiveness of sunscreens and contribute to minimize these tests in the initial phase of product research and development.
RESUMO
Introducción: La grasa infrapatelar de Hoffa es una inclusión infraarticular presente en la rodilla y existen evidencias que células multipotentes están presentes en tejido adiposo del humano adulto. Objetivo: El propósito de este trabajo es aislar e identificar células mesenquimales a partir de muestras de la grasa de Hoffa tomadas de pacientes a los que se les practicó cirugía de rodilla que expongan este tejido. Metodología: Una vez obtenido el consentimiento informado de un total de 12 pacientes, se obtuvo la muestra de tejido graso. La grasa fue disgregada incubándola en colagenasa en PBS a 37ºC y con agitación, luego se inactivó con suero fetal bovino, se separaron mediante centrifugación los adipocitos maduros de las células multipotentes y se obtuvieron las muestras para sembrar en medio Mesencult. Una porción de muestra fue utilizada para identificación mediante citometría de flujo y otra parte para ser coloreada con azul de metileno. Resultados y Discusión: Tanto en las tinciones con azul de metileno como en los cultivos celulares se han apreciado células con la morfología propia de una célula mesenquimal en forma de estrella y que fueron corroborados mediante citometría de flujo para los marcadores CD13, CD29, CD59 y CD105; igualmente hemos podido observar la efectividad del tratamiento enzimático. Conclusiones: En la actualidad nos encontramos consolidando los cultivos primarios para posteriormente diferenciarlos en líneas celulares específicas que puedan ser utilizadas en estudios de patologías, tales como obesidad, diabetes y trastornos articulares...
Introduction: The infrapatellar fat pad (sometimes know Hoffa's pad) is a soft tissue that lies beneath the patella (kneecap) separating it from the femoral condyle. There is evidence suggesting the presence of stem cells on adipose tissue in the adult human. Objective: The purpose of this work is to isolate and identify grown stem cells from Hoffa's fat samples obtained from patients undergoing surgeries exposing this tissue. Materials and Methods: After obtaining informed consent, the biopsy of Hoff's fat pad was obtained. The samples were incubated with collagenase and PBS a 37ºC and agitated, then it was inactivated with fetal bovine serum and centrifuged, washed twice with PBS, the pellet was resuspended and one part was cultivated on Mesencult medium the other part was used for flow cytometry and stained with methylene blue for morphologic analysis. Also before and after the enzyme digestion, the samples were added to 10% formaldehyde to evaluate the collagenase treatment. Results and analysis: The results show the effectiveness of the enzymatic treatment, the architecture of the adipose tissue was lost. The star shape stem cells morphology was appreciated with methylene blue in the cultures, it was corroborated by flow cytometry with CD13, CD29, CD59 and CD 105 markers. Conclusions: Primary cultures are consolidating, the next aim will be to obtain differentiated specific cell types that can be use in the study of obesity, diabetes and articular illnesses...