ABSTRACT
Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. STATEMENT OF SIGNIFICANCE: In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.
Subject(s)
Collagen , Extracellular Matrix , Cell Differentiation , Collagen/chemistry , Extracellular Matrix/metabolism , Humans , Keratinocytes , Skin , Tissue Scaffolds/chemistryABSTRACT
Skin aging is the result of superimposed intrinsic (individual) and extrinsic (e.g., UV exposure or nutrition) aging. Previous works have reported a relationship between UV irradiation and glycation in the aging process, leading, for example, to modified radical species production and the appearance of AGEs (advanced glycosylation end products) in increasing quantities, particularly glycoxidation products like pentosidine. In addition, the colocalization of AGEs and elastosis has also been observed. We first investigated the combination of the glycation reaction and UVA effects on a reconstructed skin model to explain their cumulative biological effect. We found that UVA exposure combined with glycation had the ability to intensify the response for specific markers: for example, MMP1 or MMP3 mRNA, proteases involved in extracellular matrix degradation, or proinflammatory cytokine, IL1α, protein expression. Moreover, the association of glycation and UVA irradiation is believed to promote an environment that favors the onset of an elastotic-like phenomenon: mRNA coding for elastin, elastase, and tropoelastin expression is increased. Secondly, because the damaging effects of UV radiation in vivo might be more detrimental in aged skin than in young skin due to increased accumulation of pentosidine and the exacerbation of alterations related to chronological aging, we studied the biological effect of soluble pentosidine in fibroblasts grown in monolayers. We found that pentosidine induced upregulation of CXCL2, IL8, and MMP12 mRNA expression (inflammatory and elastotic markers, respectively). Tropoelastin protein expression (elastin precursor) was also increased. In conclusion, fibroblasts in monolayers cultured with soluble pentosidine and tridimensional in vitro skin constructs exposed to the combination of AGEs and UVA promote an inflammatory state and an alteration of the dermal compartment in relation to an elastosis-like environment.
