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Validation of ultrasonography for measurement of cartilage thickness in the equine carpus

Abstract Articular cartilage thinning is an important hallmark of osteoarthritis (OA), and ultrasonography (US) is a clinically accessible tool potentially suitable for repeated evaluation. The aim of the present prospective methods comparison study was to validate US as a tool for measuring cartilage thickness in the carpus of the horse. Eight Standardbred trotters underwent US examination with 9 and 15 MHz linear transducers. Six anatomical locations in the radiocarpal joint (RCJ) and middle carpal joint (MCJ) were examined. The same joints were assessed by ultrahigh field (9.4 Tesla) magnetic resonance imaging (MRI) and histology. Associations between measurements obtained by the different modalities were assessed by ANOVA, Deming regression, Pearson correlation and Bland–Altman plots. Histologically assessed total cartilage thickness (the noncalcified cartilage (NCC) plus the calcified cartilage zone (CCZ)) overestimated thickness compared to MRI (P < 0.01) and US (P < 0.01). US 15 MHz had substantial agreement with MRI and NCC histology, and repeatability was acceptable (coefficient of variation = 8.6–17.9%) when used for assessment of cartilage thickness in the RCJ. In contrast, 9 MHz US showed poorer agreement with MRI and NCC histology, as it overestimated the thickness of thin cartilage and underestimated the thickness of thicker cartilage in the RCJ and MCJ. Moreover, repeatability was suboptimal (coefficient of variation = 10.4–26.3%). A 15 MHz transducer US is recommended for detecting changes in RCJ cartilage thickness or monitoring development over time, and it has the potential for noninvasive assessment of cartilage health in horses.


INTRODUCTION
Osteoarthritis (OA) in the carpal joint is commonly seen in Thoroughbred and Standardbred racehorses. 1,2 In racehorses, OA is thought to be a stress-related syndrome caused by repetitive overloading of the articular surface of the carpal bones. 3,4 OA is a degenerative joint disease characterized by fibrillation and gradual thinning of the articular cartilage as a consequence of joint inflammation [5][6][7], and measurement of cartilage thickness is therefore potentially a valuable tool for diagnostic and prognostication purposes. Ultrasonography (US) has great potential as a tool for measuring cartilage thickness, 8,9 however, the assessment of cartilage thickness of the equine carpus by US has never been validated.
Many imaging modalities have been used for the assessment of articular changes; nevertheless, there are several limitations that make them less useful in the detection and monitoring of OA in horses.
Radiography is cheap and available to the general clinic but does not allow direct visualization of cartilage. 10 Changes that can be seen on radiographs include joint space narrowing and collapse, enthesophytes, osteophytes, subchondral cyst-like lesions, erosion, and sclerosis. 3,10 These are changes that only develop in late-stage OA. [5][6][7] Therefore, radiography is not ideal to diagnose and monitor the early stages of OA. 10 Magnetic resonance imaging (MRI) offers noninvasive threedimensional imaging, which allows assessment of the entire joint. 3,11,12 Although MRI-based quantitative and qualitative cartilage measurement has been continuously improved for diagnosis and monitoring of the progression of OA in both human patients and horses, [12][13][14] MRI is not always available to the equine practitioner. MRI is expensive, and general anesthesia is often needed. Therefore, MRI is less attractive for clinical routine examinations, surveillance of OA progression, or serial evaluations for research purposes. 8 Compared to the abovementioned techniques, US has several advantages: high spatial resolution, 15 noninvasiveness, and no ionizing radiation exposure. 8,16 US is costeffective and easily implementable in equine clinics and hence theoretically ideal for day-to-day clinical and research-related surveillance of joint health, including measurements of cartilage thickness. 8,16 US has been shown to correlate well with MRI measurements of cartilage thickness in several human joints, including the shoulder, knee, ankle and wrist. [16][17][18][19] US has also been shown to correlate well with cartilage thickness measured by histology in the stifle of foals 20 and the human knee. 8 However, the ultrasound beam angle and accessibility of the cartilage region of interest are key determinants of the success of cartilage measurements. 21

Study design
This was a prospective, nonrandomized methods comparison study. Six were measured on sagittal images.

Histology measurements
Immediately after MRI, the carpal joints were opened by sharp dissection carefully to avoid any damage to the cartilage. Osteochondral wedge sections (5 × 5 × 10 mm) were cut out using an oscillat-

Statistical analysis
All statistical analyses were performed with a statistical software package (R, version 3.6.1, The R Foundation for Statistical Computing).
Selection and completion of statistical tests was performed by a PhD

Histological total cartilage thickness was significantly different from US and MRI measurements
An ANOVA showed that histologically measured total cartilage thickness (NCC + CCZ) was significantly thicker (1.03 ± 0.05 mm) than values obtained with US or MRI and with histologic measurements of the NCC only (US 9 MHz, 0.69 ± 0.03 mm, P < 0.001; US 15 MHz, 0.83 ± 0.04 mm, P < 0.01; MRI, 0.82 ± 0.05 mm, P < 0.01; NCC histology, 0.86 ± 0.05 mm, P < 0.01; Table 1). Therefore, histologically measured total cartilage thickness was discarded from subsequent analysis, and US was only compared to NCC histology and MRI. All cartilage thickness measurements presented as boxplots with medians, interquartile ranges, and outliers are shown in Figure 7.

Ultrasonographic cartilage thickness measurement was dependent on frequency
The same ANOVA showed that 9 MHz US measurements were significantly thinner than values obtained from all other imaging modalities (US 15 MHz, P < 0.05; MRI, P < 0.05; NCC histology, P < 0.05). In contrast, cartilage thickness assessed by 15 MHz US showed no significant difference compared to NCC histology (P = 0.725) and MRI (P = 0.858).
Additionally, there was no significant difference between NCC histology and MRI (P = 0.595; Table 1).

Association and correlation between imaging modalities
The slope of the Deming regression line of the association between US and NCC histology and between US and MRI did not differ significantly    (Figure 7), as suggested by the ANOVA (Table 1) and Bland-Altman plots (Figure 8).

Repeatability of US measurements
Particularly in the thinner cartilage of the MCJ, US 9 MHz had low precision (a high CV%) ( Table 3).
To the best of the authors' knowledge, this is the first time that US has been investigated for the assessment of carpal articular cartilage thickness in the adult horse. US has been used for the assessment of articular cartilage thickness in human patients and has been shown to correlate well with other modalities such as MRI 16,17,19 and histology. 8 Until now, US measurement of equine cartilage thickness has only been investigated in the femorotibial joint of foals. 20 In that study, cartilage thickness of the lateral and medial trochlear ridges TA B L E 3 Coefficient of variance (CV%) between three repetitions of ultrasound (US) measurements of the same anatomical location in the same horse. A CV of less than 15% was considered to represent satisfactory agreement between repetitions 18,27.  Articular cartilage is composed of the NCC and CCZ layers. The latter forms the interface between articular cartilage and subchondral bone. 31,32 Histologic measurements of total cartilage thickness, including the CCZ, were consistently larger than both measurements made by US and MRI. These findings were expected, as cartilage thickness

Joint
was measured with US as the hypoechoic band between two hyperechoic lines (Figures 3 and 4). It is generally assumed that the second hyperechoic reflection line in an US image of the osteochondral unit comes from the tidemark and hence delineates the boundary between the NCC and the CCZ. 33 The same is true for MRI sequences; because of the high mineral content, the CCZ has intrinsically short T2/T2* values, which makes it difficult to make contrast between the CCZ and underlying bone. 34 Thinning of the NCC is a hallmark of OA and is caused by both erosion and fibrillation of the superficial layer and by expansion of the CCZ into the NCC. 5  transducer. This may require improvements in image quality and correction for the nonperpendicular US beam angle. 21 Additionally, we may have been able to obtain better ultrasound images with individual optimization for each horse with regard to the angle of carpal flexion or ultrasound settings such as gain. In this study, however, we chose to standardize these conditions for enhanced repeatability. An additional limitation is related to the study population. It is possible that cartilage measurement in horses with naturally occurring carpal osteoarthritis will be more difficult because of variable lesion location, decreased range-of-motion, or loss of anatomical landmarks (e.g., erosion of the subchondral bone plate).

CONCLUSION
Cartilage thickness in the equine RCJ assessed ultrasonographically