Optical anisotropy reveals molecular order in a mouse enthesis

Abstract

Entheses are specialized biological structures that functionally anchor tendons to bones. The complexity, mechanical characteristics and properties of the entheses, particularly those related to exercise, mechanical load and pathologies, have been extensively analyzed; however, the macromolecular organization of the enthesis fibers, as assessed by polarization microscopy, has not yet been investigated. Morphological and optical anisotropy characteristics, such as birefringence, linear dichroism (LD) and differential interference contrast (DIC-PLM) properties, are thus analyzed in this study of a healthy adult mouse calcaneal tendon-bone enthesis. The molecular and supramolecular order of collagen and GAGs was determined for the collagen bundles of this enthesis. Based on a birefringence plot pattern as well as on metachromasy and linear dichroism after toluidine blue staining at pH 4.0, a similarity between the calcaneal tendon-bone enthesis and cartilage during ossification may be assumed. This similarity is assumed to favor the adequacy of this enthesis to support a compressive load. Considering that the collagen-proteoglycan complexes and the enthesis fibers themselves have a chiral nature, these structures could be acting via reciprocal signaling with the cellular environment of the enthesis.

Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells

“…Around 300 measurements were done in tendon sections of each group. With the microscope and the software it is possible to carry out analysis of DIC and anisotropic properties, both individually and in combination [56,57]. To obtain the birefringence of the fibers and collagen bundles without Walston’s prism activity, these were removed from the polarized light path by turning the condenser tower to the position of a field of observation free, giving passage only to polarized light.…”

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5

“…For the birefringence analyses of nuclei, ADMSC (5P) in culture ( n = 3) were stained with 0.025% toluidine blue solution in 0.1 M McIlvaine phosphate buffer at pH 4.3 for 30 min, followed by analyses in the Olympus BX-51 (Olympus America, Center Vallery, PA, USA) polarizing microscope equipped with a Q-color 5 camera (Olympus America), and using the Image Pro-plus v.6.3 software for Windows™ (Media Cybernetics, Silver Spring, MD, USA) [ 4 , 33 , 34 ]. With the microscope and the software, it is possible to carry out analysis of DIC and the anisotropic properties, both individually and in combination [ 34 ]. DIC observations were performed after addition of the two condenser’s Wollaston prisms in the light path.…”

“…As collagen bundles exhibit two types of birefringence, intrinsic birefringence (Bi) and form or textural birefringence (Bf) [ 38 ], the total birefringence (the sum of Bi and Bf) was used in this study. Measurements of the tendons in each experimental group were made after immersing the sections in water, a condition in which total birefringence is highly detectable [ 4 , 33 , 34 , 38 ]. The number of measurements of GA was represented as the median and they were chosen at random in 16 sections from four tendons of each group.…”

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy

“…These measurements allow distinguishing between homogenous and heterogeneous materials comprised of subdomains of molecules arranged in a particular direction. Polarization microscopy can also be used to obtain information about how the molecules within these subdomains are oriented, providing insight into the underlying molecular order of the material [9][10][11].…”