The basement membrane microenvironment directs the normalization and survival of bioengineered human skin equivalents.

Epithelial-mesenchymal interactions promote the morphogenesis and homeostasis of human skin. However, the role of the basement membrane (BM) during this process is not well-understood. To directly study how BM proteins influence epidermal differentiation, survival and growth, we developed novel 3D human skin equivalents (HSEs). These tissues were generated by growing keratinocytes at an air-liquid interface on polycarbonate membranes coated with individual matrix proteins (Type I Collagen, Type IV Collagen or fibronectin) that were placed on contracted Type I Collagen gels populated with dermal fibroblasts. We found that only keratinocytes grown on membranes coated with the BM protein Type IV Collagen showed optimal tissue architecture that was similar to control tissues grown on de-epidermalized dermis (AlloDerm) that contained intact BM. In contrast, tissues grown on proteins not found in BM, such as fibronectin and Type I Collagen, demonstrated aberrant tissue architecture that was linked to a significant elevation in apoptosis and lower levels of proliferation of basal keratinocytes. While all tissues demonstrated a normalized, linear pattern of deposition of laminin 5, tissues grown on Type IV Collagen showed elevated expression of alpha6 integrin, Type IV Collagen and Type VII Collagen, suggesting induction of BM organization. Keratinocyte differentiation (Keratin 1 and filaggrin) was not dependent on the presence of BM proteins. Thus, Type IV Collagen acts as a critical microenvironmental factor in the BM that is needed to sustain keratinocyte growth and survival and to optimize epithelial architecture.

Characterization of the initial response of engineered human skin to sulfur mustard.

We have used a new approach to identify early events in sulfur mustard-induced, cutaneous injury by exposing human, bioengineered tissues that mimic human skin to this agent to determine the morphologic, apoptotic, inflammatory, ultrastructural, and basement membrane alterations that lead to dermal-epidermal separation. We found distinct prevesication and post-vesication phases of tissue damage that were identified 6 and 24 h after sulfur mustard (SM) exposure, respectively. Prevesication (6 h) injury was restricted to small groups of basal keratinocytes that underwent apoptotic cell death independent of SM dose. Immunoreactivity for basement membrane proteins was preserved and basement membrane ultrastructure was intact 6 h after exposure. Dermal-epidermal separation was seen by the presence of microvesicles 24 h after SM exposure. This change was accompanied by the dose-dependent induction of apoptosis, focal loss of basement membrane immunoreactivity, increase in acute inflammatory cell infiltration, and ultrastructural evidence of altered basement membrane integrity. These studies provide important proof of concept that bioengineered, human skin demonstrates many alterations previously found in animal models of cutaneous SM injury. These findings further our understanding of mechanisms of SM-induced damage and can help development of new countermeasures designed to limit the morbidity and mortality caused by this chemical agent.

Predicting Cell Association of Surface-Modified Nanoparticles Using Protein Corona Structure - Activity Relationships (PCSAR).

Nanoparticles are likely to interact in real-case application scenarios with mixtures of proteins and biomolecules that will absorb onto their surface forming the so-called protein corona. Information related to the composition of the protein corona and net cell association was collected from literature for a library of surface-modified gold and silver nanoparticles. For each protein in the corona, sequence information was extracted and used to calculate physicochemical properties and statistical descriptors. Data cleaning and preprocessing techniques including statistical analysis and feature selection methods were applied to remove highly correlated, redundant and non-significant features. A weighting technique was applied to construct specific signatures that represent the corona composition for each nanoparticle. Using this basic set of protein descriptors, a new Protein Corona Structure-Activity Relationship (PCSAR) that relates net cell association with the physicochemical descriptors of the proteins that form the corona was developed and validated. The features that resulted from the feature selection were in line with already published literature, and the computational model constructed on these features had a good accuracy (R(2)LOO=0.76 and R(2)LMO(25%)=0.72) and stability, with the advantage that the fingerprints based on physicochemical descriptors were independent of the specific proteins that form the corona.