STUDY OF THE MECHANISMS TRIGGERED BY A NOVEL AMNIOTIC MEMBRANE DERIVED TREATMENT FOR SKIN INJURIES

XIMENA GUERBI; FLAVIA MICHELINI; PABLO STRINGA; LEANDRO VECCHIO; CAROLINA ZANUZZI; MATIAS ROTELA; ROCÍO COMITO; GRISELDA MORENO; ALEJANDRO BERRA

San Luis

Congreso; LXXI Reunion Anual de la Sociedad Argentina de Inmunología; 2023

Sociedad Argentina de Inmunología

Skin wound repair is a complex process balancing among innate immunityinteractions leading either to scar formation or tissue regeneration. The formercomprises patching of the wound due to quick extracellular matrix (ECM)deposition mainly by fibroblasts and might lead to a dysfunctional sealing, whilethe latter is resolved over a longer time lapse and also requires tissue architectureremodeling guided by M2 macrophages. Human amniotic membrane has beenused as a biological dressing. We found that the healing of complex skin woundshas been surprisingly improved in patients treated with a novel homogenized,lyophilized and sterilized amniotic membrane patch (hAMpe). We previouslyfound that these derivatives induced a shift in the activation profile ofmacrophages, from M1 to M2, diminished IL-6 and TNFa secretion, andincreased IL-10 and IL-8. We recently found that IL-1b secretion was alsodownmodulated in a classical way, not involving inflammasome inhibition. Theseresults emphasize the effects of hAM on the macrophage profile contributing toan anti-inflammatory background.In order to further unravel other mechanismsinvolved in tissue regeneration, we experimentally focused on fibroblasts.No cytotoxicity was found on the human cell line fibroblasts HT1080 over theevaluated range of hAM preparations, from 50 to 800 ug/ml, with a significantincrease in cell viability under 400 ug/ml stimulation (ANOVA+Tuckey test).We quantified collagen deposition in human fibroblasts from both line and primaryculture. Cell cultures were treated with hAMpe soluble components at differenttotal protein concentrations for 24 hours and after removal of culture media andPBS washing, the insoluble collagen was picrosirius red stained, and quantifiedby staining elution in alkali, which absorbs at 540 nm. Primary fibroblastconditioned with 100 ug/ml hAMpe rehydrates secreted 60% more collagen thanthe control group, and the HT1080 fibroblasts also increased in 40% its collagendeposition compared to the control group (ANOVA+ Dunnet`s test, Ⲁ=0,05).To study cell migration, we performed the wound assay. HT1080 cells showedwound closure of 4,48% for untreated fibroblasts against 22,53% for 200 ug/mlhAMpe treated condition, being significantly different with p