SKIN ELASTICITY TESTING
H
uman skin offers a protective barrier against environmental mechanical damage thanks to the reversible deformability of its structure. In
particular, the dermal extracellular matrix (ECM) ensures an essential role in skin cohesion and influences biomechanical properties of the skin. More specifically, elastic fibres represent the primary effectors of skin elasticity due to their key role in skin compliance and resilience[4,5]
.
The organised arrangement of the elastic fibre network is more important than the abundance per se of fibres, as regards the impact on the functionality of elastic fibres and thus the resulting biomechanical properties of the skin[6]
.
In vitro 3D skin models are powerful and predictive tools for the screening and efficacy testing of bioactive molecules[1]
. Such models use
artificial, synthetic or bio-based extracellular matrices to provide a cell-adhesive substrate to cell suspensions and guide the three-dimensional organisation[2]
.
Although exogenous scaffolds have played a critical role in tissue reconstruction, they create a bias when measuring the biomechanical properties of bioengineered tissues like human skin[3]
.
3D scaffold-free microtissues use the ability of cells to aggregate, adhere, proliferate, create cell-to-cell interactions, secrete their own endogenous ECM and ultimately produce their own architecture and microenvironment in a non-adhesive environment[7]
.
These bioengineered spheroid microtissues, obtained using cells only, in the absence of artificial ECM scaffolds, are increasingly used as research tools for investigating cell-matrix interactions, cell-to-cell communication, as well as for safety and efficacy testing[8-11]
.
However, such models have not been used yet to assess the biomechanical properties of skin-derived bioengineered tissues. In this study, we developed and characterised a 3D scaffold-free spheroid microtissue, exclusively composed of normal human dermal fibroblasts (NHDF) to mitigate bias in the in vitro evaluation of dermal elastic properties.
For this, we studied the elastic fibre network within microtissues cultured for eight and 15 days using two-photon autofluorescence (2PAF) imaging and we evaluated tissue stiffness at the nanoscale using atomic force microscopy (AFM). This new advanced 3D model has been successfully used to measure the efficacy of EleVastin, a novel active ingredient developed by Gattefossé, fighting against age-related loss of skin elasticity.
MATERIALS & METHODS 3D scaffold-free spheroid microtissue formation
Scaffold-free 3D spheroid microtissue grew June 2021 49
Characterisation of the biomechanical properties AFM (Bruker, Bioscope Resolve) was used to evaluate tissue stiffness at the nanoscale, as assessed by the elastic modulus, which is calculated using BioMeca Analysis software (BioMeca). A material whose Young’s modulus is high is described as rigid/stiff, whereas a material with a low Young’s modulus is said to be ‘soft’, ‘flexible’, ‘elastic’ or less stiff.
RESULTS
Structural and biomechanical characterisation of the 3D scaffold-free spheroid microtissue
The structural organisation and biomechanical properties of 3D scaffold-free spheroid microtissues, exclusively composed of NHDF, were characterised after eight and 15 days of culture using 2PAF and AFM, respectively. 2PAF can only be detected from well-organised elastic fibres with functional 3D structure. Based on 2PAF image analysis, 15-day-old spheroid microtissues were first shown to exhibit a denser, more mature elastic fibre network than eight-day- old specimens (figure 1). Indeed, the elastin content increased four-fold at D15 compared with D8 (figure 2).
Figure 1 Z projection images of elastin deposits within spheroids after eight days and 15 days of culture. Image realised by Second Harmonic Generation microscopy (objective x40, scale bar = 50µm)
ELASTIN ELASTIN
within a few days from a suspension of NHDF, which aggregate in Ultra Low Affinity plates (ULA plates, In Sphero technology). Microtissues were cultured for eight to 15 days.
Characterisation of the elastic fibre network The elastic fibre network was studied using 2PAF imaging (ZEISS, LSM880 confocal microscope), which takes advantage of the intrinsic autofluorescence capacity of elastic fibres[12]
.
Elastin autofluorescence was detected at exc. 890 nm/em. 445 nm and converted to a grey value. The quantity of elastin is directly linked to the grey value extracted by fluorescence analysis.
Spheroid at day 8
Spheroid at day 15
©Gattefossé
©Gattefossé
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