Culturing Keratinocytes on Biomimetic Substrates Facilitates Improved Epidermal Assembly In Vitro

Abstract

Mechanotransduction is defined as the ability of cells to sense mechanical stimuli from their surroundings and translate them into biochemical signals. Epidermal keratinocytes respond to mechanical cues by altering their proliferation, migration, and differentiation. In vitro cell culture, however, utilises tissue culture plastic, which is significantly stiffer than the in vivo environment. Current epidermal models fail to consider the effects of culturing keratinocytes on plastic prior to setting up three-dimensional cultures, so the impact of this non-physiological exposure on epidermal assembly is largely overlooked. In this study, primary keratinocytes cultured on plastic were compared with those grown on 4, 8, and 50 kPa stiff biomimetic hydrogels that have similar mechanical properties to skin. Our data show that keratinocytes cultured on biomimetic hydrogels exhibited major changes in cellular architecture, cell density, nuclear biomechanics, and mechanoprotein expression, such as specific Linker of Nucleoskeleton and Cytoskeleton (LINC) complex constituents. Mechanical conditioning of keratinocytes on 50 kPa biomimetic hydrogels improved the thickness and organisation of 3D epidermal models. In summary, the current study demonstrates that the effects of extracellular mechanics on keratinocyte cell biology are significant and therefore should be harnessed in skin research to ensure the successful production of physiologically relevant skin models.

Biallelic SYNE2 Missense Mutations Leading to Nesprin-2 Giant Hypo-Expression Are Associated with Intellectual Disability and Autism

“…Therefore, the effects on nesprin-2 levels in adhesive patient cells, such as in the brain, may be more pronounced due to increased pulling forces on the nesprin-2 molecules. In keratinocytes, the stiffness of the extracellular environment affects cell spreading (colony density) and has profound effects on LINC complex protein expression [ 36 ]. To gain molecular insights into the underlying regulatory mechanisms, the identification of SR5 interacting molecules, and the study of SR5 mechanobiology is essential.…”

“…Although originally viewed solely as cytoskeletal adaptor proteins, increasing experimental evidence has transformed the initial perception. Regarding the functional aspects, nesprins are known to play fundamental roles in nuclear architecture and positioning [ 14 , 15 , 16 , 17 , 18 , 19 ], directed cell migration [ 20 , 21 , 22 , 23 , 24 ], cellular signalling [ 17 , 25 , 26 ], ciliogenesis [ 27 , 28 , 29 ], and mechanobiology [ 8 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. These pivotal roles in cell biology and physiology are also evidenced in the significant phenotypes that are exhibited by nesprin transgenic and knockout murine models.…”

Layer-by-Layer Analysis of In Vitro Skin Models

“…Culturing cells on hydrogel surfaces (of approximately 50 kPa) showed increased cell density and improved the morphological thickness and organization of the epidermal layer model when compared with standard tissue culture plastic, as well as changes in mechanobiology protein expression. A suggested 50 kPa surface was recommended due to a thicker and better organized epidermal model (Figure ).…”

Stem cell‐mediated angiogenesis in skin tissue engineering and wound healing

“…Several experimental studies have clarified the ability of stem cells to differentiate into skin cells (e.g., epidermal lineages), indicating their critical role in the reconstruction of damaged skin. For instance, chemical cues and mechanical stimuli have been successfully applied to facilitate stem cells differentiation into epidermal cell lineages 6,7 …”

“…For instance, chemical cues and mechanical stimuli have been successfully applied to facilitate stem cells differentiation into epidermal cell lineages. 6,7 Still, the risk of tumorigenicity and immune rejection is among the main restriction of specific stem cell-based therapies. Moreover, there is a controversial issue on whether adult stem cells [e.g., mesenchymal stem cells (MSCs)] possess the ability to fully differentiate into endothelial cell (EC) lineages in vivo.…”