An in vitro wound healing model for evaluation of dermal substitutes

Reepithelialization of skin wounds is essential to restore barrier function and prevent infection. This process requires coordination of keratinocyte proliferation, migration, and differentiation, which may be impeded by various extrinsic and host-dependent factors. Deep, full-thickness wounds, e.g., burns, are often grafted with dermal matrices before transplantation of split-skin grafts. These dermal matrices need to be integrated in the host skin and serve as a substrate for neoepidermis formation. Systematic preclinical analysis of keratinocyte migration on established and experimental matrices has been hampered by the lack of suitable in vitro model systems. Here, we developed an in vitro full-thickness wound healing model in tissue-engineered human skin that allowed analysis of the reepithelialization process across different grafted dermal substitutes. We observed strong differences between porous and nonporous matrices, the latter being superior for reepithelialization. This finding was corroborated in rodent wound healing models. The model was optimized using lentivirus-transduced keratinocytes expressing enhanced green fluorescent protein and by the addition of human blood, which accelerated keratinocyte migration underneath the clot. Our model shows great potential for preclinical evaluation of tissue-engineered dermal substitutes in a medium-throughput format, thereby obviating the use of large numbers of experimental animals

The Effects of Human Beta-Defensins on Skin Cells in vitro

Background: Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing. Objective: We aimed to investigate the effects of human β-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration. Methods: Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation. Results: We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction. Conclusion: Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining

Modulation of activated leukocyte cell adhesion molecule-mediated invasion triggers an innate immune gene response in melanoma.

Item does not contain fulltextActivated leukocyte cell adhesion molecule (ALCAM/CD166) is a progression marker of a variety of cancers, including melanoma, and is a marker for mesenchymal stem cells. ALCAM expression triggers matrix metalloproteinase activity and correlates with the transition between superficial melanoma growth and deep dermal invasion in vivo. We previously showed that manipulating ALCAM functionality could both decrease and increase melanoma invasion, depending on the manner by which ALCAM function was altered. How ALCAM exerts these opposing invasive phenotypes remained elusive. In the present study, we analyzed differences in melanoma cell gene expression in two- and three-dimensional cultures as function of ALCAM-mediated adhesion. We identified a cluster of genes highly responsive to ALCAM functionality and relevant for melanoma invasion. This cluster is characterized by known invasion-related genes similar to L1 neuronal cell adhesion molecule and showed a remarkable induction of several innate immune genes. Unexpectedly, we identified major variations in the expression of genes related to an immunological response when modulating ALCAM function, including complement factors C1r and C1s. The expression and function of these proteinases were confirmed in protein assays and in vivo. Together, our results demonstrate a link between ALCAM functionality and the immune transcriptome, and support the assumption that ALCAM-ALCAM interactions could function as a cell signaling complex to promote melanoma tumor invasion.1 mei 201