Article Text
Abstract
Background Entheseal fibrocartilage (EF) derangement is hypothesised to be pivotal to the pathogenesis of spondyloarthritis. Ultrasound is useful for visualisation of the enthesis but its role in EF visualisation is uncertain. This work aimed to demonstrate face and content validity of ultrasound for EF visualisation both by bovine histological evaluation and EF imaging in spondyloarthritis.
Methods Achilles enthesis of 18 bovine hindfeet was visualised using a MyLab 70 ultrasound machine. The presence of tissue with EF characteristics was documented and histological confirmation was performed on five randomly selected sections using Masson trichrome staining. Ultrasound of the Achilles tendon (AT) was performed in 19 patients with spondyloarthritis and 21 healthy controls (HC).
Results The bovine EF could be visualised in all cases and seen as a thin, uncompressible, well-defined, anechoic layer between the hyperechoic bone and the hyperechoic fibrils of the enthesis both in longitudinal and transverse scans. This region corresponded to EF on histological examination. The same pattern of low signal corresponding to EF location was seen in 17/19 patients and all HC. Discontinuities of the anechoic layer around the erosions and enthesophytes were observed in the spondyloarthritis group. The thickness of the anechoic layer was not significantly different in spondyloarthritis and HC (0.5±0.1 vs 0.5±0.2 mm, p=0.9) whereas the thickness of the EF was greater in men (0.6±0.2 vs 0.5±0.1 mm; p=0.009) compared with women.
Conclusion Ultrasound can visualise EF of the AT insertion, which can be abnormal in cases of spondyloarthritis. This has implications for a better understanding of enthesopathy.
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Entheses are the insertion sites of ligaments, tendons or joint capsules into bone. Two types of entheses can be distinguished by their structure and location: fibrous and fibrocartilaginous entheses.1 2 With the advent of MRI, it has become clear that fibrocartilaginous entheses throughout the skeleton play a pivotal role in the pathogenesis of the spondyloarthropathies.2 3 A CD8 T-cell response to peptides derived from proteins present in fibrocartilage was demonstrated previously.4 In addition it was demonstrated that subchondral T-cell infiltration is dependent on the presence of cartilage at the joint surface and significantly diminishes following fibrocartilage destruction in ankylosing spondylitis.5 More recently, it has also become evident that entheseal-related disease, including fibrocartilage involvement, is also a key feature of osteoarthritis.6,–,8
Therefore the ability to image fibrocartilage could be key to a better understanding of both spondyloarthritis and osteoarthritis.
Histological studies have shown that a fibrocartilaginous enthesis is composed of four tissue components: dense fibrous connective tissue; uncalcified entheseal fibrocartilage (EF); calcified fibrocartilage and bone.1 It is the uncalcified fibrocartilage, in particular, that probably holds the key to a better understanding of the disease process in spondyloarthritis and osteoarthritis, because fibrocartilage microdamage and adjacent microscopic synovitis are common in normal aged joints.9 In studies of patients with early spondyloarthritis-related enthesitis, a macrophage infiltration of the EF was observed in the early phases of disease.10 11 Furthermore, there is a school of thought that considers the whole immunopathology of ankylosing spondylitis to be secondary to autoimmune destruction of EF.3 Therefore, it is clear that the ability to image this anatomical territory could have diagnostic implications as well as being key to an improved understanding of the disease processes involved in spondyloarthritis and osteoarthritis.
Despite the clinical significance of entheses referred to above, detailed imaging of the EF is still limited by technical issues. All components of an attachment site are ‘invisible’ with standard MRI, mainly due to short transverse relaxation times (T2). However, recently developed ultrashort echo time (UTE) pulse sequence has improved the ability of MRI to visualise the EF.12,–,16 Nevertheless, the disadvantages of MRI, including patient discomfort, lack of availability, time and cost, limit its use. Ultrasound has been increasingly used both as a diagnostic and therapy monitoring tool for enthesitis.17,–,23 Although high-resolution ultrasound provides detailed imaging of the normal enthesis structure and pathological changes including bone erosions, enthesophytes and osteophytes, we are not aware of any study that has systematically evaluated its ability to assess EF.
In this study we aimed to demonstrate the face and content validity of ultrasound to visualise EF. For this purpose, we used ultrasound for imaging EF in bovine Achilles tendons (AT) and investigated the correspondence between ultrasound and histological findings. We then showed that the same ultrasound scanning technique depicts tissue with the same characteristics in spondyloarthritis patients and in healthy individuals. Furthermore, we show imaging evidence for fibrocartilage microdamage in young subjects with spondyloarthritis and in some healthy controls (HC), which is reminiscent of histological findings from cadaveric tissue from insertions in older individuals.
Materials and methods
Ultrasound examination
Ultrasound examination was undertaken in bovine hindfoot material obtained fresh from a slaughterhouse. Each leg was cut transversely at its midpoint in order to shorten the length for a comfortable observation with ultrasound. The flexor digitalis superficialis tendon and the surrounding tissues were dissected to expose the AT and enthesis. The calcaneus was then placed upright and rigidly fixed on a surface for ultrasound imaging. Ultrasound examinations were performed on 18 specimens by using a MyLab 70 (Esaote Biomedica, Genoa, Italy), equipped with a broadband 6–18 MHz linear probe. A stand-off gel pad was used for making scanning easier. Ultrasound equipment was set in order to obtain the best contrast of the different tissues under examination, using a high gain and low dynamic range. Ultrasound examination was performed using a multiplanar scanning technique and all findings were documented in at least two perpendicular scanning plans. Particular attention was paid to ensure that the ultrasound beam direction was perpendicular to the enthesis in order to avoid anisotropy.
Histological examination
For routine histology, the region of the AT insertion was removed from five randomly selected specimens. Strips of tissue 5 mm wide were taken from the central part of the tendon insertion and fixed for 48 h in 10% neutral buffered formol saline. The specimens were decalcified in rapid decalcifier (Shandon TBD-1; Thermo Scientific, Massachusetts, USA), dehydrated with graded alcohols and embedded in paraffin wax. Serial sagittal sections (5–8 µm thick) were collected from each block and stained with H&E, Masson trichrome or Gomori's one-step trichrome.
Imaging of human tendons
Ultrasound of the AT was performed in 38 entheses of 19 patients (11 men/eight women) with spondyloarthritis (15 patients with ankylosing spondylitis, three with psoriatic arthritis and one patient with spondylitis associated with inflammatory bowel disease). All patients, except two with ankylosing spondylitis, were asymptomatic for Achilles enthesitis at the time of evaluation and eight of 19 (42%) had previous achillodynia. The age (mean value±SD) of the patients was 41.5±9.3 years and the mean duration of disease was 13.8±11.3 years. The Bath ankylosing spondylitis disease activity index and Bath ankylosing spondylitis functional index indices were 4.2±3.3 and 4.1±3.1, respectively. Forty-two Achilles enthesis of 21 HC (15 women; six men; mean age 36.8±11.1 years) who were hospital staff without any history of previous achillodynia were also assessed by ultrasound.
The ultrasound examinations were performed in a darkened room by a rheumatologist using the same equipment. The patients were asked to take a prone position with the feet hanging over the examination table in a neutral position for visualisation of the AT. Bilateral AT ultrasound examinations were carried out using a multiplanar scanning technique. Particular attention was paid to minimise the transducer pressure on the anatomical structures under examination by using plenty of gel. Care was also taken to alter the angle of probe in order to prevent the artifact known as anisotropy. The thickness of the anechoic layer was measured at its thickest point in the longitudinal scan (figure 1).
Six different subjects (three HC and three patients with psoriatic arthritis) were scanned by two ultrasonographers (RJW and SZA) using another ultrasound machine (Logiq E9; General Electric Medical Systems, Milwaukee, Wisconsin, USA, equipped with a 15–8 MHz multifrequency linear transducer) in order to investigate whether the presence of the anechoic band was machine or operator dependent.
The study was approved by the Ethical Committee of Marmara University Medical School and informed consent was obtained from all patients and controls.
Statistics
The thickness of the EF between men and women as well as spondyloarthritis patients and HC was compared by using the Mann–Whitney U test. Correlation between the thickness of the EF, body mass index (BMI) and age were investigated by Spearman's correlation analysis.
Results
Bovine studies
Ultrasound findings
In all scanned specimens, the EF could be visualised by ultrasound. The tissue was seen as a thin, uncompressible, well-defined, anechoic thin layer between the hyperechoic bone profile and the hyperechoic fibrils of the tendon (figure 2A). The EF could be seen in both longitudinal and transverse scans and was evident in all 18 cases. No data were obtained about the bovine sesamoid fibrocartilage that is situated proximal to the insertion, as an anatomical structure corresponding to the retrocalcaneal bursa in humans was not present in cows. A gross anatomical dissection of the bovine enthesis showed that the EF occupied the same anatomical territory that it does in humans (figure 2B).
Histological findings
Histological studies confirmed the presence of uncalcified fibrocartilage at the site corresponding to the shaded region in figure 2B. Masson trichrome staining showed a clear definition of the four layers of the enthesis, ie, dense fibrous connective tissue, uncalcified fibrocartilage, calcified fibrocartilage and bone (figure 2C, D). A distinct tide mark between the uncalcified and calcified fibrocartilage was present. The calcified fibrocartilage was typically less cellular than uncalcified fibrocartilage.
Human studies
The same pattern of low signal corresponding to EF location could be seen as an anechoic line over the bone profile in all HC and most patients, both in transverse and longitudinal scans (17/19 of spondyloarthritis patients and 21/21 of HC) (figure 1A). A subset of both spondyloarthritis patients and HC had discontinuities of the anechoic layer (6/17 (35.3%) vs 5/21 (23.8%); p=0.5).
Ninety-five per cent (18/19) of spondyloarthritis patients had enthesophytes and all spurs were located at the distal part of the enthesis. Five of 19 patients (26.3%) had erosions, which were all seen at the proximal part of the insertion. The ultrasound image of the EF was interrupted at all erosion sites (figure 1B). In patients with calcaneal erosions, the remaining EF over the ‘healthy’ part of the calcaneus could be visualised in four out of five patients. Two patients, in whom the anechoic layer could not be seen, had either large erosions or enthesophytes over the calcaneus, and one of them had active inflammation with a power Doppler signal inside the erosion indicating acute Achilles enthesitis. Another two patients also had focal loss of anechonecity, without any signs of bone irregularity. The thickness of the anechoic layer was not significantly different in spondyloarthritis and HC (0.5±0.1 vs 0.5±0.2 mm; respectively; p=0.9). On the other hand, the thickness of the EF was greater in men (0.6±0.2 vs 0.5±0.1 mm; p=0.009). The thickness did not correlate with BMI, age, disease duration or activity.
The anechoic layer was visualised by two observers both in HC and patients with psoriatic arthritis using a different machine. This would suggest that the finding of the anechoic band was not machine or operator dependent.
Discussion
Whether ultrasound can visualise EF has been contentious and has not been reported in a systematic way. This study for the first time provides preliminary evidence supporting the concept that ultrasound can visualise EF. We used a bovine model due to the difficulty of obtaining human EF cadaveric samples. We also demonstrated the presence of EF in normal human Achilles insertions and in patients with spondyloarthritis, in whom we confirm that disease processes including fibrocartilage attrition adjacent to sites of erosion are evident on ultrasound. Our proof of principle preliminary findings could therefore have considerable implications for imaging the basis for joint inflammation and degeneration, especially with the increasing resolution capabilities of modern scanners.
The ‘enthesis organ’ appears to be a primary target of the inflammatory response associated with spondyloarthritis and is also implicated in the pathogenesis of osteoarthritis and rheumatoid arthritis.6,–,8 24 As the fibrocartilage at insertions resists mechanical compression and stress, it is likely to be of central importance in these disease processes. Although imaging of the enthesis has improved in the past decade, there are still considerable problems in visualising EF in living patients. Ultrasound has increasingly been used both as a diagnostic tool for enthesitis and to follow up patients after treatment.18,–,22 We observed an interruption of EF in spondyloarthritis patients with erosions. EF seems to be damaged in areas close to inflammatory damage, suggesting that ultrasound may provide structural information on fibrocartilage fissuring and fibrillation that has already been demonstrated in degenerative arthritis.6 We also noted that EF damage was noted in two patients in whom erosions or enthesophytes were not evident. These observations suggest that ultrasound may be another imaging modality that might be helpful for the follow-up of inflammatory damage in spondyloarthritis patients. As most of the changes at the EF level were seen in patients who already had other bone abnormalities, it did not give additional information to other changes in most cases.
It must be pointed out that there is emergent MRI technology for the assessment of EF. Despite all regions of the enthesis being ‘invisible’ with conventional clinical MRI, there is increasing evidence that UTE sequences with echo times 20–1000 times shorter than those available on conventional clinical MRI systems can detect signals from the different short T2 tissues of entheses, and therefore visualise the enthesis.12,–,16 In a recent article, Benjamin et al14 15 showed that it is possible to image the enthesis by UTE, which has a T2 of approximately 2–10 ms, and by magic angle imaging. However, MRI is an expensive and time-consuming imaging method. It is not practical to visualise multiple entheses of different regions with MRI. Compared with the UTE MRI technique, ultrasound offers several advantages, including speed, cost, ease of access and a high resolution that could allow the visualisation of structural changes in entheses. Indeed, both techniques could prove to be complementary in the research setting.
However, this study has some limitations. When preparing the histological bovine specimen, the processes may result in a shrinking of tissue, which might complicate the direct comparison of thickness. Due to the difficulties of obtaining fresh human tissues for research, we did not perform comparable human tissue studies. Cadaver specimens could be an alternative, but such tissue may be subject to dehydration in histological preparations, which would also make accurate measurement difficult in this setting. Quantification of the findings should also be further validated regarding the low thickness of the EF and the relatively low sensitivity of ultrasound machines for measuring the thickness. This may have led to our negative results on its relation with BMI, age and disease-specific differences.
The present study provides direct validation for the use of high-resolution ultrasound to visualise EF in bovine samples and indirect validation in humans. Further work needs to be done in human/cadaveric samples and comparisons with UTE. The potential role of ultrasound for showing EF damage has implications for future research into enthesitis mechanisms in spondyloarthritis.
References
Footnotes
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Funding SZA was funded by the Articulum Fellowship.
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Competing interests None.
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Patient consent Obtained.
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Ethics approval The study was approved by the Ethical Committee of Marmara University Medical School.
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Provenance and peer review Not commissioned; externally peer reviewed.