Non-linear optical microscopy of cartilage canals in the distal femur of young pigs may reveal the cause of articular osteochondrosis

Abstract

BackgroundArticular osteochondrosis is a common cause of leg weakness in pigs and is defined as a focal delay in the endochondral ossification of the epiphysis. The first demonstrated steps in the pathogenesis consist of loss of blood supply and subsequent chondronecrosis in the epiphyseal growth cartilage. Blood vessels in cartilage are located in cartilage canals and become incorporated into the secondary ossification centre during growth. It has been hypothesized that vascular failure occurs during this incorporation process, but it is not known what predisposes a canal to fail. To obtain new information that may reveal the cause of vascular failure, the distal femur of 4 pigs aged 82–140 days was sampled and examined by non-linear optical microscopy. This novel technique was used for its ability to reveal information about collagen by second harmonic generation and cellular morphology by two-photon-excited fluorescence in thick sections without staining. The aims were to identify morphological variations between cartilage canal segments and to examine if failed cartilage canals could be followed back to the location where the blood supply ceased.ResultsThe cartilage canals were shown to vary in their content of collagen fibres (112/412 segments), and the second harmonic and fluorescence signals indicated a variation in the bundling of collagen fibrils (245/412 segments) and in the calcification (30/412 segments) of the adjacent cartilage matrix. Failed cartilage canals associated with chondronecrosis were shown to enter the epiphyseal growth cartilage from not only the secondary ossification centre, but also the attachment site of the caudal cruciate ligament.ConclusionThe variations between cartilage canal segments could potentially explain why the blood supply fails at the osteochondral junction in only a subset of the canals. Proteins linked to these variations should be examined in future genomic studies. Although incorporation can still be a major cause, it could not account for all cases of vascular failure. The role of the caudal cruciate ligament in the cause of osteochondrosis should therefore be investigated further.

Peptide Nanophotonics: From Optical Waveguiding to Precise Medicine and Multifunctional Biochips

“…Strands of high SHG intensity, indicating collagen fibers, appear to originate in the interior of the canal and align at the canal margin. Reproduced under the terms of the CC‐BY 4.0 license ( https://creativecommons.org/licenses/by/4.0/) . Copyright 2017, BioMed Central Ltd. f) SHG images of collagen alignment in malignant prostate biopsy, and its histological images of fused glands occupied by cancer cells.…”

“…[138,139] For example, SHG microscopy was applied as a cancer diagnostic tool [140,141] to probe the structural differences in the extracellular matrix of normal stroma, benign tumors, endometrioid tumors, and more. [142] Furthermore, SHG microscopy was used to identifying articular osteochondrosis in pigs, [143] and as an early diagnostic tool for corneal related diseases [144] and imaging of collagen distributions in human sclera. [145] SHG microscopy enabled to observe structural changes in optic nerve head structure [146] and for imaging skin damages.…”

“…Reproduced under the terms of the CC-BY 4.0 license (https://creativecommons.org/licenses/by/4.0/). [143] Copyright 2017, BioMed Central Ltd. f) SHG images of collagen alignment in malignant prostate biopsy, and its histological images of fused glands occupied by cancer cells. Scale bar is 100 µm.…”

Prevalence of osteochondral lesions in the fetlock and hock joints of Standardbred horses that survived bacterial infection before 6 months of age

“…Aseptic vascular failure has been associated with the process of incorporating vessels into the advancing ossification front [ 2 , 3 ]. Localisation may be further explained by variation in the quality of collagen type I fibres around individual vessels [ 40 – 42 ]. Incorporation happens a limited number of times and can therefore lead to a finite number of lesions [ 43 , 44 ].…”

Characterization of cellular and matrix alterations in the early pathogenesis of osteochondritis dissecans in pigs using second harmonic generation and two‐photon excitation fluorescence microscopy

“…In recent studies from our research group, second harmonic generation (SHG) and two photon‐excitation fluorescence (TPEF) microscopy were used to characterize normal growth cartilage and cartilage canals . These and previous studies have shown that SHG is highly sensitive and specific to collagen fibrils and that the morphology of cells could be visualized in 3D using TPEF.…”