Femoral epiphyseal cartilage matrix changes at predilection sites of equine osteochondrosis: Quantitative MRI, second-harmonic microscopy, and histological findings.

Osteochondrosis is an ischemic chondronecrosis of epiphyseal growth cartilage that results in focal failure of endochondral ossification and osteochondritis dissecans at specific sites in the epiphyses of humans and animals, including horses. The upstream events leading to the focal ischemia remain unknown. The epiphyseal growth cartilage matrix is composed of proteoglycan and collagen macromolecules and encases its vascular tree in canals. The matrix undergoes major dynamic changes in early life that could weaken it biomechanically and predispose it to focal trauma and vascular failure. Subregions in neonatal foal femoral epiphyses (n = 10 osteochondrosis predisposed; n = 6 control) were assessed for proteoglycan and collagen structure/content employing 3T quantitative MRI (3T qMRI: T1ρ and T2 maps). Site-matched validations were made with histology, immunohistochemistry, and second-harmonic microscopy. Growth cartilage T1ρ and T2 relaxation times were significantly increased (p < 0.002) within the proximal third of the trochlea, a site predisposed to osteochondrosis, when compared with other regions. However, this was observed in both control and osteochondrosis predisposed specimens. Microscopic evaluation of this region revealed an expansive area with low proteoglycan content and a hypertrophic-like appearance on second-harmonic microscopy. We speculate that this matrix structure and composition, though physiological, may weaken the epiphyseal growth cartilage biomechanically in focal regions and could enhance the risk of vascular failure with trauma leading to osteochondrosis. However, additional investigations are now required to confirm this. 3T qMRI will be useful for future non-invasive longitudinal studies to track the osteochondrosis disease trajectory in animals and humans. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1743-1752, 2016.

Differences in the vascular tree of the femoral trochlear growth cartilage at osteochondrosis-susceptible sites in foals revealed by SWI 3T MRI.

Focal ischemic chondronecrosis of epiphyseal growth cartilage (EGC) during endochondral ossification is believed to be a key early event on the pathway to osteochondrosis (OC) in both animals and humans. The lateral ridge of the equine trochlea is a site where severe osteochondritis dissecans lesions frequently arise and is a model for the study of naturally occurring disease. Non-invasive imaging to investigate EGC vascularity may help elucidate why focal ischemia occurs. 3T MRI susceptibility-weighted imaging (SWI) of femoral trochlea of OC predisposed (n = 10) and control (n = 6) day-old foals, with minimal joint loading after birth, was performed. SWI and 3D images revealed the EGC vascular architecture without a contrast agent, and matched histologic observations. No vascular lesions were identified. There was no difference in the vascular density and architecture between control and OC specimens, but a striking difference in vascular pattern was seen at the OC-predilected site in the lateral ridge of the trochlea in all specimens, when compared to the medial ridge of the trochlea, where OC lesions are rarely observed. This site was less ossified with more perichondrial vessels not yet bridging with the subchondral bone. Furthermore, the mean vascular density of all specimens was significantly higher at this site. We speculate that joint morphology and focal internal trauma on this site with a unique vascular architecture may trigger ischemic events at this site. SWI permitted visualization of EGC in young foals with a clinical 3T MRI and paves the way for non-destructive longitudinal studies to improve understanding of OC in all species. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1539-1546, 2016.

The Impact of Collagen Fibril Polarity on Second Harmonic Generation Microscopy.

In this work, we report the implementation of interferometric second harmonic generation (SHG) microscopy with femtosecond pulses. As a proof of concept, we imaged the phase distribution of SHG signal from the complex collagen architecture of juvenile equine growth cartilage. The results are analyzed in respect to numerical simulations to extract the relative orientation of collagen fibrils within the tissue. Our results reveal large domains of constant phase together with regions of quasi-random phase, which are correlated to respectively high- and low-intensity regions in the standard SHG images. A comparison with polarization-resolved SHG highlights the crucial role of relative fibril polarity in determining the SHG signal intensity. Indeed, it appears that even a well-organized noncentrosymmetric structure emits low SHG signal intensity if it has no predominant local polarity. This work illustrates how the complex architecture of noncentrosymmetric scatterers at the nanoscale governs the coherent building of SHG signal within the focal volume and is a key advance toward a complete understanding of the structural origin of SHG signals from tissues.