Comparative Medicine Copyright 2015 by the American Association for Laboratory Animal Science

Vol 65, No 4 August 2015 Pages 342–347

Original Research

Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source Glendon W Rhoades,1 George S Belev,2 L Dean Chapman,3 Sheldon P Wiebe,4 David M Cooper,3 Adelaine TF Wong,4 and Alan M Rosenberg5,* The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combined with CT and using a synchrotron radiation source. DEI–CT images of an explanted 4-wk-old piglet stifle joint were acquired by using a 40-keV beam. The series of scanned slices was later ‘stitched’ together, forming a 3D dataset. Highresolution DEI-CT images demonstrated fine detail within all joint structures and tissues. Striking detail of vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and a synchrotron radiation source can generate more detailed images of intact, growing joints than can currently available conventional imaging modalities. Abbreviation: DEI, diffraction-enhanced imaging.

Diffraction-enhanced imaging (DEI) is a biomedical imaging technique that, compared with conventional radiography, generates very detailed images with more edge contrast but deposits a lower radiation dose to the object. DEI generates enhanced contrast both from absorption, the process involved in conventional radiography, and from of X-ray refraction, a process that harnesses photons that otherwise typically are imperceptibly diffracted.4 The DEI technique collects information from X-rays that are refracted as they pass through tissues that have different refractive indices as it almost completely removes diffracted X-rays. In comparison, conventional radiography produces images from X-rays that are attenuated by the tissues through which they pass, but X-rays that are refracted within those same tissues confound, rather than clarify, image contrast. The creation of contrast from the refraction of X-rays, rather than exclusively from absorption, yields images that display more detail with clearer distinction between tissue interfaces. Refraction-based imaging can reveal tiny structures that are transparent to X-ray attenuation but have sufficient variation in density to produce refraction contrast. Furthermore, refraction-based imaging decreases the required radiation dose.21 To obviate the superimposing effects in a 2-dimensional DEI refraction image, we considered that combining CT with DEI would yield images with even greater clarity. CT allows a 3D representation of the sample, such that contrast from features Received: 06 Jan 2015. Revision requested: 19 Feb 2015. Accepted: 18 Apr 2015. Departments of 1Biomedical Engineering, 3Anatomy and Cell Biology, 4Medical Imaging, and 5Pediatrics, University of Saskatchewan, and 2Biomedical Imaging and Therapy Beamlines, Canadian Light Source, Saskatoon, Saskatchewan, Canada. * Corresponding author. Email: [email protected]

at different depths are no longer superimposed on one another but can be separated and viewed as independent structures. Although this advantage is valuable in traditional absorption imaging, the additional features that provide contrast in a refraction-based image enhance the value of CT. Combining DEI technology, which is capable of imaging soft-tissue detail, with CT, which allows segregation of the contrast images at different depths, overcomes limitations of conventional X-ray imaging, namely lack of distinction of soft tissues and 2-dimensionality. As we report here, DEI combined with CT and a synchrotron-generated X-ray source yields 3D images of growing joint tissues at a resolution on the order of micrometers, which is much higher than can be generated using conventional imaging techniques. A synchrotron radiation source was required for the development of DEI because a synchrotron currently is the only source capable of providing an intensely brilliant light (millions of times brighter than sunlight and conventional X-ray sources), is highly collimated (light rays in the beam remain parallel with negligible dispersion over distance), can be made to be monochromatic (having a single wavelength), and can be tuned precisely to an array of energy ranges. The Canadian Light Source (www.lightsource.ca), which began operations in 2005, is one of only 47 synchrotron facilities worldwide and the only such facility in Canada. Although nonsynchrotron sources of X-rays for DEI–CT are conceivable,16,18 such technology requires considerable image-acquisition time. Regardless, the quality of images generated by using synchrotron technology likely would remain the standard with which any new nonsynchrotron DEI–CT technological innovations would be compared.14

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Figure 1. (A) Axial slice of a DEI-CT refraction dataset through the femoral and tibial cartilage of a 4-wk-old porcine stifle joint. (B) Anatomic specimen, with soft tissue removed, showing the approximate orientation of the bones of the joint when imaged by DEI-CT. The vertical arrows indicate the imaging direction. (C) A lateral view of the disarticulated bones shown for orientation. The dashed box represents the approximate DEI-CT field of view. The horizontal arrow indicates the imaging direction. Dimensions, 5625 × 4219 pixels; resolution, 300 dpi.

Despite refinements in medical imaging, conventional radiography, CT scanning, and MRI still are insufficient to discern fine details, particularly in growing joints in which soft tissues (including cartilage) predominate and change with physiologic growth. The impetus for the current research was to develop an imaging technique that better demonstrated normal joint characteristics during growth and, in the future, could be applied to pathologic joints for experimental research and eventually clinical applications. In particular, we were motivated by a need to more effectively and reliably image growing joints affected by arthritis, a disease associated with alterations of bone and cartilage growth, tissue morphology and vascularity. Childhood

arthritis research likely will benefit from having an improved imaging technique to aid in early diagnosis, monitor disease progression, and assess responses to therapies. The long-term outcomes of childhood arthritis are improved with early diagnosis and prompt and effective response to treatment interventions. Clinical and laboratory-based indicators of inflammation are not always adequate to detect and monitor subclinical intraarticular inflammation which, as with overt disease, can lead to progressive joint damage. Imaging can augment clinical and laboratory assessment of arthritis activity, but even the most sensitive currently available modalities are unable to detect all joint pathology.

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In juvenile arthritis, joint-imaging outcomes are difficult to evaluate because variations associated with normal growth cannot always be easily discerned from variations induced by the disease. Conventional radiography tends to detect advanced joint damage that has affected bone, but cartilage can be assessed only indirectly, and soft tissue abnormalities cannot be fully evaluated. Consequently, conventional radiography has insufficient sensitivity and specificity to be considered useful for diagnosing or monitoring children with inflammatory joint disease.6,20 MRI, which evaluates both soft tissues and osteochondral structures, can be used to detect cartilage loss, bone erosions, and synovial hypertrophy in children and adolescents, and contrast-enhanced MRI detects active synovitis.1,10 However, standardized approaches to acquire and interpret MRI data are not established for children in general and, in particular, for children with arthritis;12,15 it is not always clear, for example, if observed thinning of cartilage is physiologic or pathologic. Furthermore, although MRI is more sensitive than conventional radiography, MRI too has limited precision in detecting fine structures and pathologic changes; a clinical MRI has less than 50% sensitivity in detecting cartilage damage that subsequently is seen arthroscopically.8,13 CT offers another option for joint visualization, given that it provides high-resolution, 3D images of bone from any angle. Despite its high spatial resolution, however, CT cannot match MRI’s soft-tissue contrast resolution, because CT provides negligible variability of attenuation coefficients of soft tissues so attenuation is nearly the same for cartilage, muscles, and ligaments. Furthermore, CT’s value is offset by the necessity for radiation exposure, a particular concern in the pediatric population. Therefore, for joint research and clinical applications, each of the conventional imaging techniques currently available has limitations. A safe, higher resolution imaging system that generates good contrast for all joint structures is required. Because the DEI technique initially was developed by using a synchrotron light source, we similarly used synchrotron technology in the current experiments. In contrast to conventional X-ray tubes, a synchrotron generates light by using radiofrequency waves and electromagnets to energize and accelerate electrons, thus producing brilliant, highly focused light from the entire wavelength spectrum, including X-rays. For the development and evaluation of DEI–CT imaging of joints, we chose to use healthy commercial piglet stifle joints because porcine stifle joints are anatomically similar to human knees.5 In addition, pigs grow quickly, reaching skeletal maturity at the distal femur and proximal tibia in 20 mo,19 thus allowing for the use of the pig as a model to study growth patterns in normal and disease states in a relatively short time period. The current study aimed to develop and test a new technology for imaging growing joints by using DEI combined with CT and a synchrotron radiation source. This report is the first to document the application of DEI–CT for imaging intact, growing joints.

Materials and Methods

Samples. This research was approved by the University of Saskatchewan’s Animal Research Ethics Board (protocol no. 20100030). For these initial proof-of-concept experiments, we chose to image stifle joints collected from 4-wk-old piglets. According to our preliminary experiments, the growth plate at this stage of development has not fused, but ossification centers have developed sufficiently that the full joint space and some trabecular

Figure 2. Z-projection through 20 axial slices of a DEI-CT refraction dataset through the femoral and tibial cartilage of a 4-wk-old porcine stifle joint, demonstrating the vascularity of the cartilage. Dimensions, 1704 × 1362 pixels; resolution, 300 dpi.

bone within the ossification centers still fit in the imaging field of view; that is, a single dataset allows visualization of joint structures including trabecular and cortical bone, epiphyseal cartilage, and ligaments, as well as surrounding muscle. Because the bones of the pig stifle joint fuse at 20 mo19 and the human knee joint bones fuses at approximately 18 y (range, 14 to 23 y [168 to 252 mo; mean, 210 mo]),17 the rate of maturation of the pig stifle joint is approximately 10 times that of the human knee joint. Thus, a 4-wk piglet stifle joint is approximately equivalent to the skeletal maturation stage of a 10-mo-old child. Hindlimbs from two 4-wk-old male piglets each weighing approximately 13 kg were provided by and housed at the Vaccine and Infectious Disease Organization (University of Saskatchewan, Saskatoon, Saskatchewan, Canada; www.vido.org) in accordance with Canadian Council on Animal Care Guidelines on the Care and Use of Farm Animals in Research, Teaching and Testing.2 Animals were euthanized by using a lethal dose of pentobarbital sodium (360 mg/kg) administered intravenously. The left stifle joint was explanted by disarticulating the hindlimb at the hip joint and severing the soft tissues. To prevent hair spicules from appearing in the images (synchrotron-based DEI–CT imaging is sufficiently sensitive to detect fine surface hairs), the skin surface was shaved. Fresh explanted joints were then mounted in a custom-manufactured clear acrylic holder to ensure reproducible stabilization during scanning. A stifle joint from one piglet was used for DEI–CT and the other for comparative imaging by using DEI without CT, plain radiography, CT without DEI, and MRI. DEI and DEI–CT protocol. Images were obtained by using the BioMedical Imaging and Therapy beamline at the Canadian Light Source synchrotron facility (University of Saskatchewan, Saskatoon, Canada), with a 40-keV beam prepared by the doublecrystal Bragg monochromator at the 4,4,0 reflection. The images were detected by using an 18.7-µm pixel image detector (XDR, Photonic Science, East Sussex, United Kingdom).21 Images were taken at 25%, 50%, and 100% reflectivity points along the analyzer rocking curve. The analyzer rocking curve is the intensity compared with angle curve as the analyzer crystal is rotated near the Bragg reflection condition. For the conditions

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Figure 3. (A) Sagittal sections approximately midjoint of DEI-CT scans of a 4-wk-old porcine stifle joint (A1; magnification, A2). The tibial ossification center is visible at the bottom of each image. Vasculature is visible within the cartilage. White arrows indicate vessels traversing through trabecular bone and bone cortex into the cartilage. (B) Sagittal z-projection of a 4-wk-old porcine stifle joint at 37.4 µm, showing vasculature in the femoral cartilage (arrows). (C) A sum-projection image shows anterior and posterior cruciate ligaments at the center of the joint. Dimensions, 2400 × 1800 pixels; resolution, 300 dpi.

chosen, this curve is approximately 2 μrad in width (1 μrad is approximately 57 millionths of a degree). Images were obtained from both the high- and low-angle sides of the rocking curve, to produce refraction and absorption images, respectively. The beam was made monochromatic through X-ray diffraction using silicon crystals. The analyzer matches the monochromator crystal and is parallel but placed after the object being imaged. A synchrotron radiation beam typically has a limited height; for example, the Biomedical Imaging and Therapy beamline produces a maximal beam height of approximately 7 mm at a 25.5-m source-to-sample distance. Consequently, scan slices were acquired sequentially by moving proximally through the joint. The series of scanned slices was later ‘stitched’ together, thus forming a 3D dataset with a much greater field of view. ImageJ software (version 1.48, NIH; http://rsb.info.nih.gov/ij/) was used to process and visualize images. The ImageJ ‘z-project’ function yields an image whose intensity is produced by showing the variance of the standard deviation of pixel intensities through a stack of slices in the dataset. To compare DEI–CT with other imaging modalities, we imaged a stifle joint from a 4-wk-old piglet by using planar DEI (that is, no CT) and conventional radiography, CT (Discover CT750 HD,

GE Healthcare, Little Chalfont, United Kingdom) and MRI (1.5 T Avanto, Siemans Healthcare, Erlangen, Germany). The purpose of this particular study was to design and test DEI–CT as a new method for imaging growing joints. To develop this technique, we used normal joints from healthy piglets. However, to begin exploring whether the technique could detect pathology, we iatrogenically induced damage to cartilage by inserting 2 acupuncture needles (diameter, 200 µm). We scanned the specimen first with the needles in place and then with the needles removed.

Results

High-resolution DEI–CT images demonstrated detail within all joint tissues (Figure 1). Cartilage edges were clear, and the cartilage-bone interface could be discerned. The cortical shell and trabecular structure of the bone were clearly displayed in the refraction images. Furthermore, the images clearly displayed ligaments within the joint. The vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints in both pigs and humans, was seen in striking detail (Figure 2).9,11 Whereas a single slice tended to give an oblique cross-sectional look at any given canal, a z-projection of the standard deviation through several

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Figure 4. Comparative nonDEI-CT images of a 4-wk-old piglet stifle joint. (A) Conventional radiography, lateral view. (B) CT, saggital view. (C) MRI, saggital view. Arrows indicate cartilage. (D) MRI, coronal view with joint partially flexed to reveal femoral and tibial cartilaginous surfaces. Arrows indicate cartilage. (E) DEI without CT, lateral view. Arrows indicate cartilage surface. Dimensions, 2716 × 726 pixels; resolution, 300 dpi.

slices permitted easy mapping of the vasculature’s path through the cartilage (Figure 3 A and B). Vascular canals traversing the cortical shell and the trabecular bone structure were seen (Figure 3 A). The canal highlighted by a double arrow in Figure 3 A had an approximate diameter of 230 µm. The image was of sufficient detail that the vascular channel was visualized. In addition, the walls of the vessel were clearly resolvable, each outlined by the signature dark–bright intensity pattern of an object of high relative density in a refraction-based dataset. Z-projection through the epiphyseal cartilage of both the tibia and femur further demonstrated the vasculature within the cartilage (Figure 3 B). Cruciate ligaments were again visible in selected slices (Figure 3 C). Resolution of DEI–CT was substantially more detailed than that obtained with other imaging modalities (Figure 4 A through E). In particular, cartilage vascularity could not be discerned with any of the other imaging methods. The 200-µm needles inserted into cartilage (Figure 5 A) and the damage they induced (Figure 5 B) could be clearly seen with DEI–CT.

Discussion

This report documents for the first time that DEI combined with CT and using a synchrotron radiation source can generate more detailed images of intact, growing joints than can other currently available conventional imaging modalities. In particular, cartilage, bone, and soft tissues and their respective interfaces and the vascularity within growing cartilage can be discerned by using this new imaging technique. Mature articular cartilage is avascular, but in humans and other mammals, including pigs, cartilage development and growth is associated with metaphyseal and epiphyseal vascularity, which is present in fetal and early postnatal life.3,7,9,11 In pigs, the epiphyseal cartilage of the femoral condyles is maximally vascularized at age 2 wk.3 By 8 to 10 wk, the axial regions of the condyles in pigs are avascular, and the abaxial regions are almost completely avascular by age 4 to 5 mo.3 Knowledge about cartilage vascularity in pediatric health and particularly in disease remains limited and rudimentary. Using the technology we have described to generate refined images in a large animal model during growth affords new opportunities to better characterize both normal and pathologic growth.

Figure 5. Damage to cartilage was iatrogenically induced by inserting 2 acupuncture needles (diameter, 200 µm) and scanning the specimen (A) with the needles (white arrows) in place and then (B) with the needles removed. One needle (arrow 1) was inserted end-on into the articular cartilage, whereas the other needle (arrow 2) was inserted transversely through the cartilage surface. Both the needles themselves (A) and the needle tracks after needle removal (B) are clearly visible. Dimensions, 1872 × 1404 pixels; resolution, 300 dpi.

In particular, studying inflammatory joint diseases that occur spontaneously due to infection or trauma or are experimentally induced in piglets should serve as a valuable ex vivo and eventual in vivo animal model to better understand pathologic processes and treatment responses in juvenile arthritis. The results we report here provide a basis and impetus for developing clinically accessible, nonsynchrotron-based DEI–CT technologies.

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Acknowledgments

This research was performed at the Canadian Light Source, which is supported, in part, by the Natural Sciences and Engineering Research Council of Canada (NSERC), the National Research Council of Canada, the Canadian Institutes of Health Research (CIHR), the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. A full list of Canadian Light Source partners can be found at http://www.lightsource.ca/about/partners/. We also acknowledge the support of the CIHR Strategic Training Initiatives in Health Research program, Training in Health Research Using Synchrotron Techniques (GR), the NSERC Discovery Grant Program (DC), the Canadian Arthritis Network, and the Pediatric Rheumatic Disease Research Laboratory (University of Saskatchewan).

References

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9. Gruber HE, Lachman RS, Rimoin DL. 1990. Quantitative histology of cartilage vascular canals in the human rib. Findings in normal neonates and children and in achondrogenesis II–hypochondrogenesis. J Anat 173:69–75. 10. Gylys-Morin VM, Graham TB, Blebea JS, Dardzinski BJ, Laor T, Johnson ND, Oestreich AE, Passo MH. 2001. Knee in early juvenile rheumatoid arthritis: MR imaging findings. Radiology 220:696–706. 11. Jaramillo D, Villegas-Medina OL, Doty DK, Rivas R, Strife K, Dwek JR, Mulkern RV, Shapiro F. 2004. Age-related vascular changes in the epiphysis, physis, and metaphysis: normal findings on gadolinium-enhanced MRI of piglets. Am J Roentgenol 182:353–360. 12. Johnson K. 2006. Imaging of juvenile idiopathic arthritis. Pediatr Radiol 36:743–758. 13. Levy AS, Lohnes J, Sculley S, LeCroy M, Garrett W. 1996. Chondral delam­ination of the knee in soccer players. Am J Sports Med 24: 634–639. 14. Li J, Wilson N, Zelazny A, Meyer J, Zhong Z, Muehleman C. 2013. Assessment of diffraction-enhanced synchrotron imaging for cartilage degeneration of the human knee joint. Clin Anat 26:621–629. 15. Miller E, Roposch A, Uleryk E, Doria AS. 2009. Juvenile idiopathic arthritis of peripheral joints: quality of reporting of diagnostic accuracy of conventional MRI. Acad Radiol 16:739–757. 16. Muehleman C, Fogarty D, Reinhart B, Tzvetkov T, Li J, Nesch I. 2010. In-laboratory diffraction-enhanced X-ray imaging for articular cartilage. Clin Anat 23:530–538. 17. O’Connor JE, Bogue C, Spence LD, Last J. 2008. A method to establish the relationship between chronological age and stage of union from radiographic assessment of epiphyseal fusion at the knee: An Irish population study. J Anat 212:198–209. 18. Parham C, Zhong Z, Connor DM, Chapman LD, Pisano ED. 2009. Design and implementation of a compact low-dose diffractionenhanced medical imaging system. Acad Radiol 16:911–917. 19. Reiland S. 1978. Morphology of osteochondrosis and sequelae in pigs. Acta Radiol Suppl 358:45–90. 20. Restrepo R, Lee EY, Babyn PS. 2013. Juvenile idiopathic arthritis: current practical imaging assessment with emphasis on magnetic resonance imaging. Radiol Clin North Am 51:703–719. 21. Wiebe S, Rhoades G, Wei Z, Rosenberg A, Belev G, Chapman D. 2013. Understanding refraction contrast using a comparision of absorption and refraction computed tomographic techniques. J Instrum 8:C05004.

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Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source.

The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combi...
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