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

Abstract

Although tissue engineered skin substitutes have demonstrated some clinical success for the treatment of chronic wounds such as diabetic and venous ulcers, persistent graft take and stability remain concerns. Current bilayered skin substitutes lack the characteristic microtopography of the dermal-epidermal junction that gives skin enhanced mechanical stability and creates cellular microniches that differentially promote keratinocyte function to form skin appendages and enhance wound healing. We developed a novel micropatterned dermal-epidermal regeneration matrix (μDERM) which incorporates this complex topography and substantially enhances epidermal morphology. Here, we describe the use of this three-dimensional (3-D) in vitro culture model to systematically evaluate different topographical geometries and to determine their relationship to keratinocyte function. We identified three distinct keratinocyte functional niches: the proliferative niche (narrow geometries), the basement membrane protein synthesis niche (wide geometries) and the putative keratinocyte stem cell niche (narrow geometries and corners). Specifically, epidermal thickness and keratinocyte proliferation is significantly (p<0.05) increased in 50 and 100 μm channels while laminin-332 deposition is significantly (p<0.05) increased in 400 μm channels compared to flat controls. Additionally, β1(bri)p63(+) keratinocytes, putative keratinocyte stem cells, preferentially cluster in channel geometries (similar to clustering observed in native skin) compared to a random distribution on flats. This study identifies specific target geometries to enhance skin regeneration and graft performance. Furthermore, these results suggest the importance of μDERM microtopography in designing the next generation of skin substitutes. Finally, we anticipate that 3-D organotypic cultures on μDERMS will provide a novel tissue engineered skin substitute for in vitro investigations of skin morphogenesis, wound healing and pathology.

Keywords: 3-D organ model; Dermal–epidermal junction; Keratinocyte function; Microtopography.

Figure 1

Production of μDERM and 3D skin model system. Photolithography was used to create a silicon wafer with microtopographic features resembling the DEJ (A–C). Polydimethylsiloxane silicone elastomer (PDMS, 10:1 base to curing agent; Sylgard184; Dow Corning) was cured on the wafer’s surface creating a negative mold (D). Type I collagen was self-assembled on the micropatterned molds (E) and a collagen-GAG sponge was laminated to the collagen matrix, and crosslinked to form the μDERM (F). μDERMs were conjugated with fibronectin (FN, 32 μg/cm²; BD Biosciences) and sterilized (G). The dermal side was seeded with fibroblasts and for 48 hours (H) then the micropatterned epidermal surface was seeded with keratinocytes (I). After 48 hours, μDERMs were cultured at the air-liquid interface. (J) Photograph of PDMS pattern used to create μDERMs. The collagen gel was cast over the entire area of the patterns, which were designed to fit into 6 well plates. (K) μDERM (collagen gel side up) on cell seeding screen in a 6 well plate. After removal from patterns, μDERMs were trimmed to 1.25cm × 2.0 cm and placed on cell-seeding screens. (L) Side view of μDERM on cell-seeding screen. Screens enable air-liquid interface culture. (M) Low magnification image of μDERM after 7 days of air-liquid culture.

Figure 2

Hematoxylin and eosin stain of cultured μDERMs. Keratinocytes cultured on μDERM form a stratified epidermis. μDERMs containing fibroblasts exhibit epidermal organization at 3 days (A, D, G, J). At 7 days, epidermal thickness is increased and distinct basal, spinous, granular, and cornified layers are observed (B, E, H, K). At both 3 and 7 days, μDERMs containing fibroblasts resemble DED controls (P, Q) and 7 day cultures are morphologically similar to neonatal foreskin controls (R). Keratinocytes cultured on flats (M, N) stratify, but lack a defined stratum granulosum present in microtopographies. Additionally, epidermal thickness is increased by channel topographies. In contrast, μDERMs cultured without fibroblasts (C, F, I, L, O) are characterized by a disrupted basal and suprabasal layers as well as poor epidermal stratification and differentiation. Scale bar = 100μm.

Figure 3. Morphometric analyses of epidermal thickness in keratinocyte microniches

Epidermal thickness (ET) is increased in narrow channel topographies. Epidermal thickness was normalized by dividing by channel depth (CD); three measurements were taken per well and averaged (A). Normalized epidermal thickness (C) increased from days 3 to 7 and by co-culture with fibroblasts, independent of topography. Additionally, normalized epidermal thickness in narrow channels is statistically increased compared to wider channels cultured under the same conditions. This difference is most pronounced on μDERMs cocultured with fibroblasts. Plateau thickness (PT) was measured immediately adjacent to each channel topography (B) and in the center of control flats. Plateau thickness increased adjacent to narrow channels and decreased adjacent to wide channels relative to flats (D). Data presented as mean±SEM. †indicates statistical difference between groups, p<0.05. * indicates statistical difference between indicated channel dimensions, p<0.05. Scale bar = 100μm.

Figure 4. Proliferation on μDERM

Representative images of ki67 expression on μDERMs with 50μm (A), 100μm (B), 200μm (C), and 400μm (D) channels, on flat μDERM (E) and on DED (F). Dotted white line indicates DEJ. The linear density of ki67+ keratinocytes was calculated by normalizing against DEJ length (G) and planar graft length (H). Ki67+ cell density along the length of the DEJ within 100 μm channels is increased compared to 200 μm channels, 400 μm channels and flats. Ki67+ cell density along the length of the graft is significantly increased in regions containing 50 μm channels compared to 200 μm channels, 400 μm channels, and flats. Additionally, the graft density of ki67+ cells in narrow channeled regions is not statistically different from native foreskin. Scale bar = 100μm. Data presented as Mean ±SEM. * denotes statistical differences between specified microniche dimensions, ¥ indicates statistical difference from foreskin control, p<0.05.

Figure 5. Basement membrane protein deposition on μDERM

Representative images of laminin-332 expression (Alexafluor546) on cultured μDERMs with 50μm (A), 100μm (B), 200μm (C), and 400μm (D) channels, on flat μDERM (E) and on DED (F). LUT false colorization of laminin 332 expression (G–L); blue = highest 1/3 laminin γ2 expression, green = mid 1/3 laminin γ2 expression, red = lowest 1/3 laminin γ2 expression. Laminin deposition was quantified by normalizing the relative fluorescent intensity (RFI) of 2 expression at the DEJ against DEJ length (M). Laminin 332 deposition is increased in wider channels and decreased in narrow channels compared to flat control features. There was no statistical difference in regional expression of laminin-332 within microniches (plateaus, channel base, channel sides) of a specific dimension. Data presented as mean ±SEM. c. Asterisks indicate statistical difference from: *50 μm channels, **100μm channels, *** 200μm channels, ****400μm channels. ¥ Indicates statistical difference from flats control, p<0.05.

Figure 6. Cellular Localization on μDERM

LUT false colorization of β₁ integrin and p63 expression with 50μm (A), 100μm (B), 200μm (C), and 400μm (D) channels, on flat μDERM (E) and on DED (F); blue = highest 1/3 β₁ expression, green = mid 1/3 β₁ expression, red = lowest 1/3 β₁ expression, white = p63⁺ nuclei. Insets show indicated regions of interest at high magnification. β₁^brip63⁺ keratinocytes preferentially localize to the bases of 50μm (A) and 100μm (B) channels and the corners of 400μm channels (D). In contrast, β₁^bri cells are randomly distributed on flat regions and p63 expression is limited. On DED, β₁^brip63⁺ cells are located in niches (F). Scale bar = 100μm.