Development of Microfabricated Dermal Epidermal Regenerative Matrices to Evaluate the Role of Cellular Microenvironments on Epidermal Morphogenesis

Abstract

Topographic features at the dermal-epidermal junction (DEJ) provide instructive cues critical for modulating keratinocyte functions and enhancing the overall architecture and organization of skin. This interdigitated interface conforms to a series of rete ridges and papillary projections on the dermis that provides three-dimensional (3D) cellular microenvironments as well as structural stability between the dermal and epidermal layers during mechanical loading. The dimensions of these cellular microenvironments exhibit regional differences on the surface of the body, and quantitative histological analyses have shown that localization of highly proliferative keratinocytes also varies, according to the regional geometries of these microenvironments. In this study, we combined photolithography, collagen processing, and biochemical conjugation techniques to create microfabricated dermal epidermal regeneration matrices (mDERMs) with features that mimic the native 3D cellular microenvironment at the DEJ. We used this model system to study the effect of the 3D cellular microenvironment on epithelialization and basal keratinocyte interaction with the microenvironment on the surface of the mDERMs. We found that features closely mimicking those in high-friction areas of the body (deep, narrow channels) epithelialized faster than features mimicking low-friction areas. Additionally, when evaluating b1 expression, an integrin involved in epidermal morphogenesis, it was found that integrin-bright expression was localized in the depths of the features, suggesting that the mDERMs may play a role in defining cellular microenvironments as well as a protective environment for the regenerative population of keratinocytes. The outcomes of this study suggest that mDERMs can serve as a robust biomimetic model system to evaluate the roles of the 3D microenvironment on enhancing the regenerative capacity and structural stability of bioengineered skin substitutes.

Micropatterned dermal–epidermal regeneration matrices create functional niches that enhance epidermal morphogenesis

“…μDERM fabrication was modified from a previous method [28] by incorporating fibroblasts into the dermal sponge and decreasing the thickness of the collagen gel-matrix (Figure 1). Specifically, a pattern was designed with a series of 200μm deep channels with varying widths (50μm, 100μm, 200μm, 400μm) [28, 29].…”

“…Specifically, a pattern was designed with a series of 200μm deep channels with varying widths (50μm, 100μm, 200μm, 400μm) [28, 29]. Type I acid-soluble collagen (10 mg/ml in 5 mM HCl) was purified from rat tail tendons [30, 31] and 0.3 ml of this collagen solution was self-assembled on the micropatterned molds using 5x DMEM (Invitrogen) with 0.22 M NaHCO 3 and 0.1 M NaOH (Sigma) for 18 hours at 37°C and 10% CO 2 .…”

“…μDERMs were inverted onto A/L screens, NHKs were seeded on the micropatterned side at a density of 5×10 5 cells/cm 2 in 100μl seeding medium for 2 hours then μDERMs were submerged in seeding medium(Figure 1I). As a control, de-epidermized dermis (DED) was seeded with keratinocytes and cultured alongside μDERMs, as previously described [28]. After 24 hours, seeding medium was replaced with priming medium for 48 hours.…”

“…We recently described a method to create micropatterned dermal-epidermal regeneration matrices (μDERMs) that facilitate the investigation of cellular responses to microtopographies [28]. In this study, we integrated fibroblast co-culture into our model system and specifically probed the formation of distinct proliferative and synthetic niches within micropatterned keratinocyte microniches.…”

Evaluation of a bilayered, micropatterned hydrogel dressing for full-thickness wound healing

“…Pins et al developed microfabricated dermal-epidermal regeneration matrices comprised of 50 mm wide channels and showed that these synthetic 3D microenvironments may indeed create a protective environment for a regenerative population of keratinocytes similar to that of native skin tissue. 17,60 Based on these findings, the advantage of the Sharklet-micropatterned apical layer on the bilayered dressing evaluated here may be twofold: (1) accelerated epidermal cell migration and (2) guided development of appropriate keratinocyte niches at the dermal-epidermal junction.…”