High-Performance Polarization Microscopy Reveals Structural Remodeling in Rat Calcaneal Tendons Cultivated In Vitro.

Collagenous tissues exhibit anisotropic optical properties such as birefringence and linear dichroism (LD) as a result of their structurally oriented supraorganization from the nanometer level to the collagen bundle scale. Changes in macromolecular order and in aggregational states can be evaluated in tendon collagen bundles using polarization microscopy. Because there are no reports on the status of the macromolecular organization in tendon explants, the objective of this work was to evaluate the birefringence and LD characteristics of collagen bundles in rat calcaneal tendons cultivated in vitro on substrates that differ in their mechanical stiffness (plastic vs. glass) while accompanying the expected occurrence of cell migration from these structures. Tendon explants from adult male Wistar rats were cultivated for 8 and 12 days on borosilicate glass coverslips (n = 3) and on nonpyrogenic polystyrene plastic dishes (n = 4) and were compared with tendons not cultivated in vitro (n = 3). Birefringence was investigated in unstained tendon sections using high-performance polarization microscopy and image analysis. LD was studied under polarized light in tendon sections stained with the dichroic dyes Ponceau SS and toluidine blue at pH 4.0 to evaluate the orientation of proteins and acid glycosaminoglycans (GAG) macromolecules, respectively. Structural remodeling characterized by the reduction in the macromolecular orientation, aggregation and alignment of collagen bundles, based on decreased average gray values concerned with birefringence intensity, LD and morphological changes, was detected especially in the tendon explants cultivated on the plastic substrate. These changes may have facilitated cell migration from the lateral regions of the explants to the substrates, an event that was observed earlier and more intensely upon tissue cultivation on the plastic substrate. The axial alignment of the migrating cells relative to the explant, which occurred with increased cultivation times, may be due to the mechanosensitive nature of the tenocytes. Collagen fibers possibly played a role as a signal source to cells, a hypothesis that requires further investigation, including studies on the dynamics of cell membrane receptors and cytoskeletal organization, and collagen shearing electrical properties.

Skin collagen fiber molecular order: a pattern of distributional fiber orientation as assessed by optical anisotropy and image analysis.

BACKGROUND: Birefringence can reveal much of the morphology, molecular order, heterogeneity of fiber orientation, and nonlinear optical properties of biopolymers such as collagen. However, the detailed characterization of skin collagen fibers using optical anisotropy methods remains elusive. A clear understanding of collagen fiber organization in skin tissues may be important in the interpretation of their structural-functional relationships under normal and pathological conditions. In this study, fiber orientation in collagen bundles (CBs) and their supramolecular organization were examined in rat skin using polarization microscopy and image analysis. METHODOLOGY/PRINCIPAL FINDINGS: Image variations with rotation of the microscope stage and selection of the in-depth focus plane were investigated in unstained sections of varying thicknesses from rat skin fragments. Total birefringence (image analysis) and form and intrinsic birefringence (Sénarmont's method) were estimated. Based on the birefringent images, CBs were found to contain intercrossing points with a twisted helical distribution of collagen fibers (chiral elements) and frequently presented circular structures. Collagen fibers were observed to extend from the surface level to deeper planes, creating a 3D-network of oriented intertwined CBs. At least three levels of birefringent brilliance intensity were revealed by image analysis, indicating a heterogeneous spatial organization of the CBs. Slight differences in optical retardations were found for CBs immersed in some of the fluids used in a comparison of 170- and 240-day old rats. CONCLUSION/SIGNIFICANCE: Polarization microscopy studies provide detailed high-quality structural information on rat skin CBs. A 3D-network structure based on image analysis and birefringence compensation for collagen fibers is suggested for CBs. Form and intrinsic birefringence evaluation can reveal differences in the rat skin associated with age at the levels of collagen fiber crystallinity and macromolecular organization. These findings may inspire future studies of the feedback mechanisms by which spatial, bioelectrical and biomechanical information is transmitted from CBs to skin cells.