TESTING SKIN ELASTICITY
Figure 2 Analyses of the Second Harmonic Generation images. Mean elastin fluorescence signal quantification (grey level) within spheroids after eight days and 15 days of culture
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106.76 x4
Figure 3 Distribution of elastic modulus extracted from spheroids at eight days and 15 days of culture. Student test: *** p-value <0,0005
25.56 Spheroid at day 8 Spheroid at day 15
Then, we showed that stiffness was also significantly decreased over culture time. The mean apparent stiffness decreased from 500 kPa for microtissues at D8 to 250 kPa for microtissues at D15 (figure 3). Moreover, 2PAF analysis combined with AFM allowed us to draw a correlation map of apparent stiffness and elastin deposit density, which revealed that elastin-rich areas were less stiff (figure 4).
This confirmed that the elastic properties of 3D scaffold-free spheroid microtissues correlate with the amount of newly formed elastic fibres. Taken together, our results highlighted that the elastic tissue within 3D scaffold-free spheroid microtissues forms a sufficiently extensive network to contribute to the dermal mechanics. The correlative study outlined 3D scaffold-free spheroid microtissues as a reliable micro-dermis model to evaluate the efficacy of active ingredients displaying properties against age-related loss of skin elasticity.
DISCUSSION
The essential functions of the human skin depend on the mechanical properties of the dermis, which provides elasticity and resistance to stretch.
Changes in dermal mechanics occur during intrinsic ageing, photodamage, hypertrophic scarring and fibrosis[6]
.
In addition, mechanical alterations of the skin tissue are mainly due to ECM collapse. Indeed, the ECM provides biomechanical cues regulating vital processing regarding cell behaviour (adhesion, differentiation, proliferation, migration and even survival) and further tissue formation[13,14]
.
Consequently, residing fibroblasts adapt the stiffness of their cytoskeleton to that of their substrate[15,16]
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As a result, understanding tissue homeostasis requires an appreciation of cell and tissue mechanical properties and this approach is an absolute necessity when designing in vitro reconstructed dermis models[17]
.
In tissue engineering and regenerative medicine, most 3D scaffold models use artificial extracellular matrices to encourage cell attachment and modulate cell behaviour. ECM- mimicking biomaterials aim to restore dynamics, composition and structure of the native ECM[18]
.
Collagen-based scaffolds comprise a large majority of artificially made skin substitutes currently available for clinical use. The major drawback when using collagen as the main component of scaffolds for skin substitutes is its relatively poor mechanical strength[19]
.
Conversely, skin substitutes which use exogenous polymers to increase mechanical strength, decrease their cellular biological responses[20]
.
Considering that elastic fibres in native dermis are the primary effectors of elastic recoil following mechanical deformation[6]
, we chose to
develop 3D scaffold-free spheroid microtissues in which both elastic fibres’ synthesis and mechanical properties are ensured. The structural organisation of these spherical microtissues enhanced dermal fibroblast interaction and further allowed them to secrete their own ECM, which form a complex 3D microenvironment close to dermis in vivo. 2PAF image analysis of spheroid microtissues allowed us to observe the 3D macrostructure of elastin fibres thanks to their autofluorescence capacity[12]
.
Fifteen-day-old spheroid microtissues exhibited a denser, more mature elastic fibre network than an eight-day-old specimen. This highlighted that a longer culture time of fibroblasts is a necessary condition for optimal secretion and assembly of extracellular matrix macromolecules such as elastin.
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Spheroid at day 8 Spheroid at day 15
Mean elastin fluorescence signal (U.A)
Apparent stiffness (kPa)
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