Abstract
The junction between the epidermal and dermal layers of human skin undulates, the width and depth of the undulations varying with age and disease. We previously showed that when primary human epidermal keratinocytes are seeded on collagen-coated undulating static polydimethylsiloxane (PDMS) elastomer substrates, the stem cells, differentiated cells, and proliferating cells become patterned in response to cues from the underlying substrate. To investigate how patterning occurs over time, we have now created a dynamic model, in which a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane is placed over a polyimide sheet containing circular holes, differing in diameter and spacing. When a vacuum is applied the membrane is induced to undulate, the heights of the undulations depending on the pressure applied and the size of the holes. We observed clustering of cells with high levels of β1 integrin expression, a stem cell marker, in the base of the undulations within 48 h of applying the vacuum. Differentiating involucrin-positive cells did not cluster; however, there was clustering of cells with high E-cadherin expression and nuclear YAP. Rho kinase inhibition resulted in loss of clustering, suggesting a role for Rho family members in the process.Impact StatementIn human skin the junction between the epidermis and dermis undulates. Epidermal stem cells pattern according to their position relative to those undulations. Here we describe a rig in which epidermal cells are cultured on a collagen-coated poly(d,l-lactide-co-glycolide) (PLGA) membrane. When a vacuum is applied the membrane is induced to undulate. Stem cells cluster in response to the vacuum, whereas differentiating cells do not. Rho kinase inhibition results in loss of clustering, suggesting a role for Rho family members in the process. This dynamic platform is a new tool for investigating changes in the skin with age and disease.
Highlights
Mammalian skin is a complex organ that consists of two major layers, the epidermis and the dermis, separated by a basement membrane that is rich in type IV collagen and laminin.[1]
Using a panel of undulating collagen-coated PDMS substrates[18] that differ in diameter, height, and center-to-center spacing, we have previously shown that topography is sufficient to direct the formation of b1 integrin bright stem cell clusters on the top of the features
The polymer concentration of each membrane was chosen based on the minimum concentration that could create an elastic thin membrane that would deform under vacuum pressure without collapsing: 2% (w/v) PLGA and 5% (w/v) PLA
Summary
Mammalian skin is a complex organ that consists of two major layers, the epidermis and the dermis, separated by a basement membrane that is rich in type IV collagen and laminin.[1]. Dermal junction, in most body sites clustering on the top of the dermal papillae.[9,11,12] Expression of several cell surface markers is enriched in human epidermal stem cells, including b1-integrin receptors, Lrig[1], CD46, MCSP, and Delta-like 1. Other approaches include culture on deepidermized acellular human dermal matrices,[15] self-assembled living sheets made with human fibroblasts and keratinocytes,[16] and bioprinted cell-laden hydrogels.[17] these models fail to mimic the undulating epidermal–dermal junction. To overcome this limitation several other models[18,19,20] have been developed. Undulations have been created using static topographies through microfabrication of dermal–epidermal regeneration matrices, for example, through fabrication of patterned polydimethylsiloxane (PDMS) substrates or micro-topographies using photolithography followed by the production of collagenGAG templates.[19]
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