Characterization and quantification of in-vitro equine bone resorption in 3D using µCT and deep learning-aided feature segmentation.

High cyclic strains induce formation of microcracks in bone, triggering targeted bone remodeling, which entails osteoclastic resorption. Racehorse bone is an ideal model for studying the effects of high-intensity loading, as it is subject to focal formation of microcracks and subsequent bone resorption. The volume of resorption in vitro is considered a direct indicator of osteoclast activity but indirect 2D measurements are used more often. Our objective was to develop an accurate, high-throughput method to quantify equine osteoclast resorption volume in µCT 3D images. Here, equine osteoclasts were cultured on equine bone slices and imaged with µCT pre- and postculture. Individual resorption events were then isolated and analyzed in 3D. Modal volume, maximum depth, and aspect ratio of resorption events were calculated. A convolutional neural network (CNN U-Net-like) was subsequently trained to identify resorption events on post-culture µCT images alone, without the need for pre-culture imaging, using archival bone slices with known resorption areas and paired CTX-I biomarker levels in culture media. 3D resorption volume measurements strongly correlated with both the CTX-I levels (p < 0.001) and area measurements (p < 0.001). Our 3D analysis shows that the shapes of resorption events form a continuous spectrum, rather than previously reported pit and trench categories. With more extensive resorption, shapes of increasing complexity appear, although simpler resorption cavity morphologies (small, rounded) remain most common, in acord with the left-hand limit paradigm. Finally, we show that 2D measurements of in vitro osteoclastic resorption are a robust and reliable proxy.

An in vitro model for discovery of osteoclast specific biomarkers towards identification of racehorses at risk for catastrophic fractures.

BACKGROUND: Focal bone microcracks with osteoclast recruitment and bone lysis, may reduce fracture resistance in racehorses. As current imaging does not detect all horses at risk for fracture, the discovery of novel serum biomarkers of bone resorption or osteoclast activity could potentially address this unmet clinical need. The biology of equine osteoclasts on their natural substrate, equine bone, has never been studied in vitro and may permit identification of specific biomarkers of their activity. OBJECTIVES: (1) Establish osteoclast cultures on equine bone, (2) Measure biomarkers (tartrate resistant acid phosphatase isoform 5b [TRACP-5b] and C-terminal telopeptide of type I collagen [CTX-I]) in vitro and (3) Study the effects of inflammation. STUDY DESIGN: In vitro experiments. METHODS: Haematopoietic stem cells, from five equine sternal bone marrow aspirates, were differentiated into osteoclasts and cultured either alone or on equine bone slices, with or without a pro-inflammatory stimulus (IL-1ß or LPS). CTX-I and TRACP-5b were immunoassayed in the media. Osteoclast numbers and bone resorption area were assessed. RESULTS: TRACP-5b increased over time in osteoclast cultures without bone (p < 0.0001) and correlated with osteoclast number (r = 0.63, p < 0.001). CTX-I and TRACP-5b increased with time for cultures with bone (p = 0.002; p = 0.02 respectively), correlated with each other (r = 0.64, p < 0.002) and correlated with bone resorption (r = 0.85, p < 0.001; r = 0.82, p < 0.001 respectively). Inflammation had no measurable effects. MAIN LIMITATIONS: Specimen numbers limited. CONCLUSIONS: Equine osteoclasts were successfully cultured on equine bone slices and their bone resorption quantified. TRACP-5b was shown to be a biomarker of equine osteoclast number and bone resorption for the first time; CTX-I was also confirmed to be a biomarker of equine bone resorption in vitro. This robust equine specific in vitro assay will help the study of osteoclast biology.

Bilateral thoracic radiographs increase lesion detection in horses with pneumonia or pulmonary neoplasia but do not bring any additional benefit for inflammatory or diffuse pulmonary disease.

Published studies describing the effects of bilateral radiographic projections on the detection of equine pulmonary lesions are currently lacking. The objectives of this retrospective, single center, observational study were to compare unilateral and bilateral thoracic radiographic projections for the detection of pulmonary lesions in a group of horses. Based on their clinical diagnosis, 167 adults and foals with bilateral thoracic radiographs were classified as having pneumonia (n = 88), inflammatory or diffuse pulmonary disease (n = 72), and pulmonary masses (n = 7). After an initial interrater repeatability test, right-to-left and left-to-right projections were anonymized and independently interpreted by a radiologist blinded to the clinical diagnosis. Scores were attributed for each pattern/lesion (alveolar, interstitial, bronchial, nodules/masses, cavitary lesions) and each quadrant. Agreement between scores from each projection was evaluated with Bland-Altman plots. Lesions identified on one side but not on the contralateral projection were considered discordant. There was no preferential lateralization of pulmonary lesions. The prevalence of discordance was 14.4%, 9.0%, and 4.2% for alveolar pattern, nodules/masses, and cavitary lesions, respectively. Up to nine horses (10.2%) with pneumonia could have been misdiagnosed. A pulmonary mass would have been missed in one case. For inflammatory or diffuse disease, discordance was slight, and the addition of contralateral projections had no impact on radiographic interpretation. In conclusion, in horses with pneumonia or neoplasia, bilateral projections, or adding at least one contralateral caudoventral view, increased the probability of identifying pulmonary lesions. In horses with inflammatory or diffuse disease, bilateral thoracic radiography provided no additional benefit.