Collagen fibril organization within rat vertebral bone modified with metastatic involvement

Abstract

Metastatic involvement diminishes the mechanical integrity of vertebral bone, however its specific impact on the structural characteristics of a primary constituent of bone tissue, the collagen-I fibril matrix, has not been adequately characterized. Female athymic rats were inoculated with HeLa or Ace-1 cancer cells lines producing osteolytic or mixed (osteolytic & osteoblastic) metastases respectively. A maximum of 21days was allowed between inoculation and rat sacrifice for vertebrae extraction. Linear polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and transmission electron microscopy (TEM) imaging was utilized to assess the impact of metastatic involvement on collagen fibril organization. Increased observations of deviations in the typical plywood motif or a parallel packing structure and an increased average measured susceptibility ratio (related to relative degree of in-plane vs. out-plane fibrils in the analyzed tissue area) in bone adjacent to metastatic involvement was indicative of change in fibrilar organization compared to healthy controls. In particular, collagen-I fibrils in tumour-induced osteoblastic bone growth showed no adherence to the plywood motif or parallel packing structure seen in healthy lamellar bone, exhibiting a much higher susceptibility ratio and degree of fibril disorder. Negative correlations were established between measured susceptibility ratios and the hardness and modulus of metastatic bone tissue assessed in a previous study. Characterizing modifications in tissue level properties is key in defining bone quality in the presence of metastatic disease and their potential impact on material behaviour.

Second Harmonic Generation of Unpolarized Light

“…Consistent with previous reports [3–5,15] for z -cut quartz and collagen far from resonance, Eq. (3) can be simplified by making the approximation of | χ z ′ x ′ x ′ | ≅ | χ x ′ x ′ z ′ | and rewritten with two parameters: the azimuthal rotation angle ϕ and a defined ratio ρ ≡ χ z ′ z ′ z ′ / χ z ′ x ′ x ′ .…”

“…The results shown in Fig. 4 are largely insensitive to the particular assumed value of ρ l , yielding qualitatively similar results when assuming ρ l = 1.4 (consistent with other reports for the local-frame tensor element ratio) [4,15]. …”

“…A per-pixel fit was performed to recover a map of polar tilt angle θ with the assumption of a local-frame hyperpolarizability ratio ρ l = 1.7 based on previously reported values in which the range of the polar tilt angle at each pixel was determined experimentally [4,15]. This value of the local-frame ratio for collagen fibers is quite similar to the most probable ratio observed experimentally [Fig.…”

“…Barzda and co-workers have introduced the Stokes-Mueller framework for the theoretical description of nonlinear optical polarimetry based on a “super-Mueller matrix” approach originally developed by Mclain and Shi [12–14]. This approach has been used to study crystalline and collagen fibril organization for polarization dependent measurements [15,16]. However, a large number of observables with many different incident polarization states are required in order to populate all 36 unique elements of the super-Mueller matrix.…”

Collagen chirality and three‐dimensional orientation studied with polarimetric second‐harmonic generation microscopy

“…Studying the ultrastructural changes in collagen assembly contributed to understanding developmental processes as well as disease progression and regeneration processes. Hence, collagen has been actively studied over the past few decades using different techniques such as transmission electron microscopy (TEM) and atomic force microscopy (AFM) in order to investigate its structural and mechanical properties as well as its function in various tissues and environments.…”