Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction

Abstract

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.

Dehydroabietic Acid Induces Regeneration of Collagen Fibers in Ultraviolet B-Irradiated Human Dermal Fibroblasts and Skin Equivalents

“…To prepare collagen matrix gels containing NHDFn, we followed the previously reported protocol [11] with some modification. Briefly, 200 µL of complete cell culture media containing 1 × 10 5 NHDFn, 7.5 µL of 1 N sodium peroxide, and 100 µL of 6 mg/mL type I bovine collagen solution (#5010, Advanced Biomatrix) per well was premixed and added on a 24-well plate.…”

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…In particular, SHG microscopy has been demonstrated extremely powerful to image collagen rich tissues [65] such as cornea [66,67], tendon [68,69], and arteries [70]. In particular, SHG microscopy has been mainly used for selectively investigating collagen fibres orientation and their structural changes in human dermis [71][72][73][74][75], keloid [76][77][78], fibrosis [79][80][81], thermally-treated samples [82][83][84][85][86], and also in tumour microenvironments [87][88][89][90][91][92][93]. In fact, SHG microscopy highlights morphologic changes in collagen structure, which indicate particular disease states, such as tumour invasiveness, as well as indicators of collagen remodelling in tumour stroma, which is playing a key-role in the tumour development from in-situ to invasive stage.…”

Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

“…SHG is particularly powerful in providing background-free imaging of collagen 39,50 in various tissues such as cornea, 51,52 tendon, 53,54 and arteries. 55 SHG microscopy has already been used for investigating structural organization of collagen in human dermis, [56][57][58][59][60] keloid, [61][62][63] brosis, [64][65][66] thermally treated samples, [67][68][69][70][71] and also in tumor microenvironment. [72][73][74][75][76][77][78] SHG microscopy can be used to detect altered physiological conditions in various tissues, including muscle, 79,80 bones 81,82 and cartilages.…”