Collagen glycation versus chronologically-aged fibroblasts in keratinocyte differentiation of reconstructed human skin

Skin morphology markedly changes over the lifespan, but the effects on the skin barrier function remain to be elucidated for nanocarrier-enhanced and –targeted topical drug delivery [1]. Herein, we investigated the effects... [ view full abstract ]

Skin morphology markedly changes over the lifespan, but the effects on the skin barrier function remain to be elucidated for nanocarrier-enhanced and –targeted topical drug delivery [1]. Herein, we investigated the effects of collagen glycation and fibroblast age, respectively, on keratinocyte differentiation in reconstructed human skin (RHS).
For glycation, collagen was incubated with 10 mM D-ribose for 3 weeks, dialyzed in water for 48 h, and analyzed by fluorescence spectroscopy [2]. Glycated collagen was mixed with non-glycated collagen 1:1 and used for RHS. Juvenile keratinocytes were used for the epidermal compartment of all RHS. Fibroblasts for glycated and juvenile (non-glycated) RHS were from juvenile foreskin; too, in addition we used fibroblasts from mammary reduction surgeries of 60- to 70-year-old female donors. RHS were cultured for two weeks after the exposure to the air-liquid-interface and then subjected to reflectance confocal microscopy, immunofluorescence staining or skin surface pH analysis.
We detected 30% of collagen glycation by fluorescence spectroscopy and significant amounts of carboxymethyl lysine staining in glycated models. Stratum corneum thickness increased in glycated and aged RHS 2-fold compared to juvenile RHS with the most dense stratum corneum appearance in glycated RHS. The surface pH of glycated models exceeded the surface pH of RHS with chronologically-aged fibroblasts and juvenile RHS. Expression of keratinocyte terminal differentiation markers like filaggrin and loricrin as well as integrin expression was enhanced in glycated RHS whilst dermal thickness decreased in glycated models compared to juvenile and aged RHS (Figure 1), which is in accordance to literature [3].
Taken together, both approaches mimic hallmarks of cutaneous aging. Collagen glycation affects keratinocyte differentiation more extensively than the replacement of juvenile by chronologically-aged fibroblasts, highlighting the impact of glycation for the skin barrier function. Future studies will evaluate the functionality of the skin barrier.

References
[1] Biniek et al. (2015). J Dermatol Sci 80: 94-101.
[2] Pennacchi et al. (2015). Tissue Eng Part A 21:2417-25.
[3] Pageon et al. (2015). BioResearch Open Access, 4.1.