Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction.
Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.
L'Oréal Research and Innovation, 1 Avenue Eugene Schueller BP22, 93601 Aulnay Sous Bois, France. ampena@rd.loreal.com
Ana-Maria
AM
Pena
Dominique
D
Fagot
Christian
C
Olive
Jean-François
JF
Michelet
Jean-Baptiste
JB
Galey
Frédéric
F
Leroy
Emmanuel
E
Beaurepaire
Jean-Louis
JL
Martin
Anne
A
Colonna
Marie-Claire
MC
Schanne-Klein
D000577:Amides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D024022:Fibrillar Collagens / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D055620:Optical Phenomena; D011725:Pyridines / Q000494:pharmacology; D012867:Skin / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D023822:Tissue Engineering; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors
D000577:Amides; D004791:Enzyme Inhibitors; D024022:Fibrillar Collagens; D011725:Pyridines; C066737:RHOA protein, human; C108830:Y 27632; D020742:rhoA GTP-Binding Protein