Computed tomography of cartilage and meniscus using anionic and cationic contrast agents

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Natural Sciences

ATIONS | JUUSO HONKANEN | COMPUTED TOMOGRAPHY OF CARTILAGE AND MENISC

Contrast enhanced computed tomography (CECT) is a promising technique for detecting articular

cartilage pathologies. However, its potential for meniscal imaging has not been thoroughly

studied. CECT could enable simultaneous quantitative imaging of these tissues provided

that the differences between their diffusion kinematics were known. CECT using anionic

and cationic contrast agents was utilized to study articular cartilage and meniscus and the differences between their diffusion kinematics.

JUUSO HONKANEN

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JUUSO HONKANEN

Computed Tomography of Cartilage and Meniscus

Using Anionic and

Cationic Contrast Agents

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 230

Academic Dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the SN201 in Snellmania Building at the University of Eastern

Finland, Kuopio, on 21^stJune 2016, at 12:00 o’clock.

Department of Applied Physics

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Grano Oy Jyväskylä, 2016

Editors: Prof. Jukka Tuomela, Prof. Pertti Pasanen Prof. Pekka Toivanen, Prof. Matti Vornanen

Distribution:

University of Eastern Finland Library / Sales of publications P.O. Box 107, FI-80101 Joensuu, Finland

tel. +358-50-3058396 http://www.uef.fi/kirjasto

ISBN: 978-952-61-2156-7 (printed) ISSNL: 1798-5668

ISSN: 1798-5668 ISBN: 978-952-61-2157-4 (pdf)

ISSN: 1798-5676 (pdf)

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Author’s address: University of Eastern Finland Department of Applied Physics P.O.Box 1627

70211 Kuopio Finland

email: juuso.honkanen@uef.fi / jtj.honkanen@gmail.com Supervisors: Professor Juha Töyräs, Ph.D.

University of Eastern Finland Department of Applied Physics Kuopio, Finland

email: juha.toyras@uef.fi Dean Jukka Jurvelin, Ph.D.

email: jukka.jurvelin@uef.fi

Adjunct Professor Tuomas Virén, Ph.D.

email: tuomas.viren@uef.fi

Reviewers: Associate Professor Martin Englund, M.D., Ph.D.

Lund University

Department of Clinical Sciences Lund, Sweden

email: martin.englund@med.lu.se Docent Mika Kortesniemi, Ph.D.

Helsinki University Hospital Medical Imaging Center Helsinki, Finland

email: mika.kortesniemi@hus.fi

Opponent: Associate Professor Marc Levenston, Ph.D.

Stanford University

Department of Mechanical Engineering Stanford, CA, United States

email: levenston@stanford.edu

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ABSTRACT

Osteoarthritis (OA) is the most prevalent joint disease, causing joint pain and dysfunction. OA affects the whole joint and it is usually characterized by a progressive loss of the articular cartilage. In early OA, the cartilage surface starts to fibrillate, the proteoglycan (PG) content decreases and the water content increases. OA can be initiated by a focal cartilage trauma, but it may also be consequence of meniscal injury or degeneration as the diminished load bearing capability exposes cartilage to excessive strains. If these pathologies are diagnosed early enough, cartilage and meniscus damage may be repaired and the further progression of tissue degeneration prevented. However, the detection of minor lesions and early degenerative changes is challenging or impossible with traditional radiological methods.

Contrast enhanced computed tomography (CECT) is a promising imaging technique for detecting cartilage pathologies. The technique relies on the diffusion of contrast agents that enables further assessment of morphological and structural properties of cartilage.

Indeed, CECT has shown potential at detecting PG loss in articular cartilage in vitro. In addition, the technique has produced promising results in ex vivo and in vivo imaging of cartilage. However, the feasibility of exploiting CECT in meniscal imaging has not been thoroughly investigated. Furthermore, the differences between the diffusion kinematics of these two tissues are unknown. In this thesis, the differences in contrast agent diffusion in articular cartilage and meniscus are studied.

First, the feasibility of utilizing CECT to image the diffusion of anionic contrast agent (ioxaglate) in isolated human meniscus was evaluated with a clinical cone beam CT (CBCT) in situ. Next, the diffusion kinematics of two different contrast agents, a small molecular anionic iodine and novel cationic contrast agent bearing two positive charges (CA2+), were determined in two independent laboratory studies. In both studies, the diffusion kinematics were determined in bovine articular cartilage and meniscus. Finally, the

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potential of CECT using anionic ioxaglate to quantitatively assess the compositional, histological and biomechanical characteristics of articular cartilage in an intact cadaveric human knee joint were evaluated using a clinical CBCT scanner.

The diffusion of ioxaglate required over 25 hours to reach diffusion equilibrium within intact human meniscus. However, the contrast agent partition at the 40 minute time point correlated significantly (p < 0.05) with that at the 30 hour time point. Further- more, the contrast agent partition after 30 hours of diffusion agreed qualitatively with the distribution of PGs. Even though the time needed for ioxaglate to reach equilibrium is not feasible for clinical applications, the significant relationship between early and late time point contrast agent partition suggests that meniscal pathologies could possibly be detected even after short, clinically-relevant, diffusion times (e.g. < 2 h). The contrast agent partition at diffusion equilibrium was significantly higher (p< 0.05) in the meniscus than in cartilage when anionic iodine was used but significantly higher (p < 0.05) in cartilage when CA2+ was used. The distribution of the contrast agent at equilibrium was different in cartilage and meniscus with both contrast agents. These results suggest that the contrast agent distribution in the meniscus may be less dependent on the PG distribution compared with cartilage. These differences in diffusion kinematics between cartilage and meniscus should be acknowledged when interpreting CECT images of knee joint. Differences in cadaveric cartilage quality between intra-joint regions were quantitatively distinguishable shortly after ioxaglate exposure as well as at diffusion equilibrium. Furthermore, X-ray attenuation correlated significantly (p< 0.05) with histological, biochemical and biomechanical properties at both early (< 2 hours) and later time points. These results suggest that CECT enables the assessment of biochemical and biomechanical properties of cartilage at clinically-relevant diffusion times. Thus, CECT may provide a useful quantitative tool for the clinical detection of the early signs of cartilage degeneration.

To conclude, the diffusion characteristics of contrast agents vary

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between articular cartilage and meniscus. Nevertheless, CECT could provide a clinical tool for simultaneous assessment of both cartilage and meniscus, as long as the limitations of the technique and differences between the tissues are understood and considered while interpreting the images.

National Library of Medicine Classification: QT 36, WN 160, WN 206, WE 300, WE 870

Medical Subject Headings: Diagnostic Imaging; Tomography, X-Ray Computed; Contrast Media; Ioxaglic Acid; Iodine; Diffusion; Carti- lage, Articular; Menisci, Tibial; Knee Joint; Osteoarthritis/diagnosis Yleinen suomalainen asiasanasto: kuvantaminen; tomografia; kontrasti;

varjoainetutkimus; diffuusio; nivelrusto; polven nivelkierukat; nivel- rikko

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Preface

This study was carried out during the years 2012-2016 in the De- partment of Applied Physics at the University of Eastern Finland.

First, I would like express my sincere gratitude to all my supervisors for their professional guidance during this Ph.D. thesis. I am grateful to my principal supervisor Professor Juha Töyräs, Ph.D., for his enthusiasm, work ethics and encouragement. He has been the main driving force behind my research. I thank my second supervisor, Dean Jukka Jurvelin, Ph.D., for his endless support and always making me see the bigger picture. I also thank my third supervisor, Adjunct Professor Tuomas Virén, Ph.D., for his help, especially, at the beginning of my thesis project.

The official reviewers of this thesis, Associate Professor Martin Englund, M.D., Ph.D, and Docent Mika Kortesniemi, Ph.D, are acknowledged for their constructive criticism and professional review.

I am grateful to Ewen MacDonald, D.Pharm., for the linguistic review.

I would like to thank my co-authors for their significant contri- butions. Especially, I would like to thank Mikael Turunen for his immeasurable help with the second and third study.

Special thanks to Lasse Räsänen and Petri Tanska for their in- valuable help and support as well as sharing this experience start- ing from the very first lecture at the university. Without you, I prob- ably would not be writing this. I would also thank Harri Kokkonen for his help over the years.

I owe my sincere thanks to the colleagues in BBC (Biophysics of Bone and Cartilage) group. It has been a privilege working in a such high spirited research group. Special thanks to Markus, Pete, Lasse, Jaakko, Anni, Kata, Satu, Katariina, Mika, Mikael, Janne, Jarkko, Moukku, Hande, Pikku Jukka, Pia, Weiwei, Rambo Roimela, Chuby, Elvis, Ari, Sami, Harri, Finski, Simo, Cristina, Roope, Jari, Mikko, Kimmo, Mimmi and Nissi. I also thank all the researchers who

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have helped me with my thesis.

I would like to express my gratitude to all my friends outside the academic world for supporting me and providing me with a full life outside of science. I thank Black Sabbath for enriching my life with all the music and introducing me to the world of Rock and Roll. Furthermore, I would like to thank my bandmates Alain Aquamelon, Bruce Deluxe, Barry Oyster and S. McDee (Thermate).

This thesis work was financially supported by the Academy of Finland (project 269315), Instrumentarium Science Foundation, Kuopio University Hospital (EVO/VTR 5041715, 5041731, 5041746 and 5063535), Magnus Ehrnrooth Foundation, National Doctoral Programme of Musculoskeletal Disorders and Biomaterials (TBDP), Orion Corporation Research Foundation, Saastamoinen Founda- tion, Sigrid Juselius Foundation and strategic funding of the Uni- versity of Eastern Finland.

I am sincerely grateful to my parents, Merja and Raimo, and my little sister Saara. Mum and dad, thank you for encouraging me in my studies and supporting my choices throughout my life. Your support means a lot to me. I would like to express my deepest gratitude to my beloved Miitu for her love, support and understanding for the long working hours.

Jyväskylä, May 2016

Juuso Honkanen

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ABBREVIATIONS

2D Two-dimensional

3D Three-dimensional

BLOKS Boston-Leeds Osteoarthritis Knee Score CA2+ Contrast agent bearing two positive charges CA4+ Contrast agent bearing four positive charges CBCT Cone beam computed tomography

CECT Contrast enhanced computed tomography

CT Computed tomography

CTa Computed tomography arthrography DD Digital densitometry

dGEMRIC Delayed gadolinium enhanced magnetic resonance imaging of cartilage

ECM Extracellular matrix

EDTA Ethylenediaminetetraacetic acid disodium salt FCD Fixed charge density

FTIR Fourier transform infrared spectroscopy GAG Glycosaminoglycan

ICRS International Cartilage Repair Society

NaI Sodium iodide

NCP non-collagenous protein microCT X-ray microtomography MRI Magnetic resonance imaging OA Osteoarthritis

OARSI Osteoarthritis Research Society International OD Optical density

PBS Phosphate buffered saline

PG Proteoglycan

PTOA Post-traumatic osteoarthritis SD Standard deviation

SNR Signal-to-noise ratio

WORMS Whole Organ Magnetic Resonance Imaging Score w.w. Wet weight

ZnSe Zinc-selenide

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SYMBOLS AND NOTATIONS [. . .] Concentration

C Solute concentration C¯ Bulk concentration d Diameter

d_sd Source-to-detector distance dso Source-to-object distance D Diffusion coefficient e Napier’s constant Eeq Equilibrium modulus E_i Instantaneous modulus F Faraday constant h Thickness of the tissue I Intensity

I₀ Initial intensity J Diffusion flux

M Magnification or molar mass µ Linear attenuation coefficient n Number of samples

ν Poisson’s ratio O Original object size O⁰ Magnified object size

p Level of statistical significance Ψ Membrane potential

q Electric charge

r Donnan ratio or Pearson correlation coefficient R Gas constant

ρ Spearman’s rho

t Time

T Temperature x Distance

z Valence of the ion Z Atomic number

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LIST OF PUBLICATIONS

This thesis consists of the present review of the author’s work in the field of contrast enhanced computed tomography of cartilage and meniscus and the following selection of the author’s publications:

I Honkanen J.T.J., Danso E.K., Suomalainen J.-S., Tiitu V., Jur- velin J.S., Töyräs J., “Contrast enhanced imaging of human meniscus using cone beam CT”. Osteoarthritis and Cartilage, 23(8), 1367–76 (2015).

II Honkanen J.T.J., Turunen M.J., Tiitu V., Jurvelin J.S., Töyräs J.,

“Transport of iodine is different in cartilage and meniscus”.

Annals of Biomedical Engineering. In press (2016).

III Honkanen J.T.J., Turunen M.J., Saarakkala S., Grinstaff M.W., Ylärinne J.H., Jurvelin J.S., Töyräs J., “Cationic contrast agent diffusion differs between cartilage and meniscus”. Annals of Biomedical Engineering. In press (2016).

IV Stewart R.C.*, Honkanen J.T.J.*, Kokkonen H.T., Tiitu V., Saa- rakkala S., Joukainen A., Snyder B.D., Jurvelin J.S., Grinstaff M.W., Töyräs J., “Contrast-enhanced computed tomography enables quantitative evaluation of tissue properties at intra- joint regions in cadaveric knee cartilage”. Submitted (2016).

(*Equal contribution)

Throughout the thesis, these papers will be referred to by Roman numerals.

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AUTHOR’S CONTRIBUTION

The publications in this dissertation are original research papers on contrast enhanced computed tomography (CECT) of articular cartilage and meniscus. The author was involved in the planning and design of each paper.

In paper I, the author conducted CECT and MRI imaging of the menisci and carried out all the data analysis excluding those of the biomechanical measurements. The biomechanical measurements were conducted and the results analysed by E. K. Danso. The author was the principal author of the manuscript.

In paper II, the author conducted or supervised the CECT mea- surements, carried out all the data analysis, and was the principal author of the manuscript.

In paper III, the author conducted or supervised the CECT mea- surements, conducted the digital densitometry, carried out all the data analysis, and was the principal author of the manuscript.

In paperIV, the author conducted the CECT imaging, biomechanical measurements and data analyses in equal contribution with R.

C. Stewart. In addition, the author conducted the histological grading of the samples with J. Töyräs and V. Tiitu. The manuscript was written in equal contribution with R. C. Stewart.

In all papers, the collaboration with the co-authors has been significant.

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1 Introduction

Articular cartilage and meniscus are soft tissue structures crucial for the proper functioning of a healthy knee joint. Articular cartilage is an avascular, highly specialized connective tissue that covers the ends of articulating bones. The crescent shaped menisci are mostly avascular fibrocartilaginous tissues located between the medial and lateral femoral condyles and tibial plateaus. These hydrated soft tissues with an inhomogeneous structure and unique mechanical properties enable near-frictionless joint movement, distribute loads in the knee joint and absorb mechanical energy during movements [1–8]. In addition, the menisci stabilize the knee joint and contribute to proprioception [2, 9].

While the structural constituents are similar in cartilage and meniscus, their contents and organization differ considerably [2, 7, 10]. Their main structural constituents are water, collagen and proteoglycans (PGs). Any disturbances in these constituents, e.g., due to inflammation or trauma, may affect the interactions between these structures, and thus impair the function of the tissues. The disrupted functioning of cartilage or meniscus may lead to the development of a degenerative joint disease called osteoarthritis (OA).

OA is the most prevalent joint disease, causing joint pain and dysfunction [4,11]. It is a disease of the whole joint, usually characterized by a progressive loss of the articular cartilage accompanied by remodeling and sclerosis of subchondral bone. The cartilage degeneration is usually caused by a disturbance in the natural repair cycle of cartilage [12], however, the pathogenesis of OA is not fully understood. Age is one of the most important risk factors for OA. Indeed, it has been estimated that over 50% of people over 50 years, and nearly 90% of people at the age of 75 years have radiological changes typical of OA [11]. Other recognized risk factors include genetics, sex (higher risk for women), congenital anomalies and obesity [4, 13–15]. In addition to these risk factors, OA may

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also be initiated by a local or global joint trauma; in such cases the disease is called post-traumatic OA (PTOA). PTOA may be initiated after a trauma to cartilage itself or a meniscal injury,e.g., a meniscal tear [16].

Conventional radiography is the gold standard imaging technique for OA diagnostics [17]. However, it can only detect changes related to moderate- to late-stage OA, such as alterations in bone and joint space narrowing, as the soft tissues (e.g., cartilage and meniscus) are not visible on plain radiographs. Magnetic resonance imaging (MRI) provides good soft tissue contrast and sufficient in-plane resolution for cartilage and meniscus assessment.

However, MRI lacks rapid three-dimensional (3D) data acquisition with a small enough isotropic voxel size - a feature that limits the detection of incipient cartilage or meniscal changes. Furthermore, the high imaging costs and long queuing times restrict the routine use of the MRI in the diagnostics of early stage cartilage and meniscal changes. Nevertheless, early diagnosis of cartilage and meniscal injuries or degeneration is essential for an effective treatment and slowing the progression of tissue degradation. More impor- tantly, if these injuries are detected early enough, the progression of the tissue degeneration might be stopped or even reversed with repair surgery or pharmaceuticals [18–20]. Thus, this lack of a sensitive imaging technique represents lost opportunities for clinicians to guide treatment before the damage has become too widespread or severe, with no other treatment options but arthroplasty.

The earliest degenerative changes in cartilage include the loss of PGs, structural changes in superficial collage, and an increase in the water content [21, 22]. Delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC) and contrast enhanced computed tomography (CECT) have been introduced for the detection of this PG loss in cartilage [23–28]. These techniques are based on an assumption that the partitioning of mobile anionic contrast agents into cartilage is inversely proportional to the tissue’s anionic fixed charge density (FCD) created by the glycosaminogly- cans (GAGs) of PGs. In addition to FCD, the diffusion and distri-

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Introduction

bution of the contrast agent are influenced by the water content, collagen content, and the structural integrity of the tissue [29–32].

Recently, these techniques have also been applied for imaging the meniscus [33–36].

Cationic contrast agents have recently been introduced for CECT imaging of cartilage [37–39] and meniscus [35]. In contrast to traditional anionic contrast agents, cationic contrast agents are assumed to distribute at diffusion equilibrium in concentration directly proportional to the FCD distribution in the tissue. Furthermore, as the cationic contrast agent molecules are attracted by the negative FCD, cationic contrast agents are assumed to offer more sensitive technique for direct monitoring of changes in the PG contents of cartilage and meniscus [35, 37].

Although many in vitro studies have highlighted the potential of CECT to assess the health of cartilage [24, 25, 28, 40–42], the feasibility of CECT for quantitative imaging of meniscus has not been thoroughly studied. Furthermore, if these two soft tissues in the knee joint could be imaged and assessed simultaneously, CECT could provide a method for comprehensive evaluation of the knee joint health with a short acquisition time and high isotropic resolution. This thesis focuses on the assessment of differences in contrast agent diffusion in cartilage and meniscus. In study I, the poten- tial of CECT to image contrast agent diffusion in human meniscus with a clinical cone beam CT (CBCT) is evaluated. The diffusion kinematics of a small anionic contrast agent (iodine) is determined in bovine cartilage and meniscus in study II while the diffusion kinematics of a novel cationic contrast agent bearing two positive charges (CA2+) in these tissues is determined in study III. Study IV examines whether CBCT based on an anionic contrast agent, reflects the biochemical, histological and biomechanical characteristics of articular cartilage in an intact cadaveric human knee joint.

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2 Knee joint

The knee joint (Figure 2.1) is the largest joint in the human body. It consists of two articulations: one between the femur and tibia, and the other between the patella and the patellar surface of the femur.

In addition to these three bones, the knee joint contains articular cartilage, ligaments, menisci, muscles and tendons. The shape of the joint does not provide as much stability as in other major joints e.g. the hip joint, but the stability of the knee is maintained by the shape of the condyles and menisci in combination with ligaments, joint capsule and surrounding muscles [43].

Figure 2.1: Illustration of human knee joint anatomy in coronal (left) and sagittal (right) plane.

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2.1 ARTICULAR CARTILAGE

Articular cartilage is a highly specialized connective tissue. The hyaline cartilaginous tissue covers the ends of articulating bones in the diarthrodial joints. Articular cartilage has a pearly blue and translucent appearance. In a human knee joint, the mean cartilage thickness varies between 2 and 4 mm depending on the anatom- ical location [44]. It allows near-frictionless movement of the articulating bones in conjunction with the synovial fluid and meniscus [8, 45]. In addition, cartilage and menisci absorb kinetic energy and distribute loads minimizing the peak stresses on underlying subchondral bone [3, 10].

2.1.1 Structure and composition of articular cartilage

The majority of articular cartilage comprises the extracellular matrix (ECM) which surrounds the cartilage cells, chondrocytes (Fig- ure 2.2). The ECM consists mainly of water (60-85% of the wet mass [7, 8]) and the water content decreases towards the cartilage- bone interface [3, 46]. In addition to water, this interstitial fluid contains small proteins, metabolites, and a high concentration of cations to balance the negative FCD of the PGs [3]. The majority of the water can flow freely in and out of the cartilage; this flow of water plays an important role not only in the tissue’s mechanical function but also in providing nutrition to the chondrocytes [47].

As cartilage is avascular, the transport of nutrients occur primarily via diffusion and convection from the synovial fluid.

Most of the solid phase of cartilage consists of collagen, which accounts for 10-22% of the wet weight of cartilage [7, 8]. In healthy cartilage, type II collagen is abundant, representing 90-95% of the collagen present in ECM [3, 46]. Other collagen types include types III, VI, IX, X, XI, XII, and XIV [48]. The collagen fibres form a tight three dimensional arcade-like network that provides high tensile and shear strength properties as well as dynamic compressive stiffness on articular cartilage [3, 48–50].

Articular cartilage is usually divided into three layers based

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Knee joint

on the orientation of the collagen fibres: superficial or tangential zone, middle or transitional zone, and deep or radial zone [51].

Beneath the deep zone is calcified cartilage which anchors the cartilage into the subchondral bone [52, 53]. In the superficial zone, the collagen fibres are oriented in parallel to the articulating surface. Even though this zone is thinnest (~4-10% of the cartilage thickness [51, 54–56]), it makes a major contribution to the tensile strength of cartilage [3]. Collagen fibres are oriented more randomly in the middle zone and perpendicular to the articulating surface in the deep zone, which is also the thickest zone (~60-85%

of cartilage thickness [51, 54, 57]). The extensive cross-linking of the collagen fibrils stabilizes the network and improves the tensile stiffness [46, 48].

PGs are complex macromolecules that are composed of a protein core with covalently attached GAG chains [7, 58]. The PG monomers form large aggregates with long hyaluronic acid chains to which they are bound from one end [8, 52]. These aggregates are entrapped and immobilized in the collagen network. PGs account for 5-10% of the wet weight of cartilage, and the PG concentration increases with tissue depth (Figure 2.2) [7, 59]. The sulphate (-SO4) and carboxyl (-COOH) groups of GAG molecules carry a negative charge, creating a negative fixed charge within the tissue [60]. The negative fixed charge attracts cations and water into cartilage, re- sulting in the swelling of the tissue. This is constrained by the collagen matrix, generating a swelling pressure that contributes to the mechanical properties of cartilage [61, 62].

In addition to collagen and PGs, ECM includes a small amount of several different non-collagenous proteins (NCPs) as well as chondrocytes. NCPs help to anchor the chondrocytes to collagen fibrils as well as contributing to organizing and maintaining the macro- molecular structure of ECM [46, 63]. Chondrocytes are highly differentiated cells that are responsible for the synthesis and main- tenance of the ECM of cartilage [46, 64]. Articular cartilage is relatively acellular; the chondrocytes account only for about 1-2% of the total tissue volume [65, 66]. The shape, size and number of chon-

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drocytes varies as a function of tissue depth. In the superficial zone, chondrocytes are smaller, flatter and are arranged in parallel to the articular surface [3, 46]. They generally have a greater density and number in the superficial zone than deeper in the matrix [46, 67].

Chondrocytes in the deep zone are more spheroidal in shape and tend to align into columns perpendicular to the articular surface [3].

Figure 2.2: Illustration of the structure of articular cartilage (left) and proteoglycan aggre- gate (right). The collagen fibres are oriented in parallel to the articulating surface while the orientation is random in the middle zone and perpendicular to the surface in the deep zone.

The intensity of the red color indicates the proteoglycan concentration in healthy articular cartilage, i.e. the proteoglycan concentration increases with tissue depth. Proteoglycan monomers are composed of a core protein to which negatively charged glycosaminoglycan chains are attached. Furthermore, the monomers are bound from one end to a hyaluronic acid chain.

2.1.2 Mechanical properties of articular cartilage

Articular cartilage has to withstand great compressive, tensional and shear stresses caused by joint loading. The ability of articular cartilage to sustain these high stresses is attributable to its multi- phasic nature [61, 68]. Due to the viscoelastic properties of cartilage (i.e., it exhibits both viscous and elastic characteristics under

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Knee joint

deformation), it exhibits a time-dependent behaviour under loading [10]. During dynamic loading, the low permeability of cartilage resists the water flow and the collagen network restricts the deformation [10, 61]. This increases the pressure within cartilage, result- ing in a high stiffness of cartilage during high-rate loading [10]. In case of a static loading, the interstitial water flows slowly out of the tissue and into the less pressurized areas. This softens cartilage and allows the distribution of loads to larger areas. This deformation continues until an equilibrium between the load and the forces resisting it within the matrix is reached. At this point, PGs are predominantly responsible for supporting the load [10, 69]. Once the load is released, the water flows back into the tissue due to the os- motic pressure created by the FCD, and the cartilage swells back to its original shape.

Besides distributing loads and decreasing stresses on subchondral bone, articular cartilage provides a wear-resistant, low friction surface for joint movement. This is achieved by lubrication by the macro-molecules (e.g. lubricin) within the synovial fluid [70]. In addition, a thin film decreasing the friction is formed on top of the articulating surfaces during dynamic loading as the pressurized water flows out of the cartilage [71].

2.2 MENISCUS

The menisci of the knee joint are two fibrocartilaginous crescent- shaped discs found between the femoral condyles and tibial plateaus. Although they were earlier thought to be vestigial remnants of a leg muscle [72], menisci are important structures within the knee joint. They redistribute contact forces across the joint, absorb shock, increase stability of tibiofemoral articulation, and aid in joint lubrication and proprioception [2, 7, 73, 74]. They are wedge-shaped in cross-section and partially cover the femoral and tibial joint surfaces occupying approximately 70% of the total contact area of the joint [75, 76].

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2.2.1 Anatomy

The medial and lateral menisci differ in shape and size; the medial meniscus is approximately 3 cm wide (from the attachment of the posterior horn to the outermost edge of the peripheral rim) and 4-5 cm long while the lateral meniscus is 3 cm wide and 3-4 cm long (Figure 2.3) [77]. The medial meniscus is C-shaped and its posterior horn is substantially larger than the anterior horn. The lateral meniscus is more circular and regular in its width.

The circumferential collagen fibres of the meniscus continue into the anterior and posterior insertional ligaments, which attaches the menisci to the tibia at their anterior and posterior horns [78]. The menisci are also attached by several other secondary ligaments and to the inside of the joint capsule at their peripheral rims [9, 79]. In addition, the medial meniscus is attached more firmly at its mid- point to deep medial collateral ligament [79]. For to this reason, the medial meniscus is less mobile that its lateral counterpart.

2.2.2 Structure and composition of meniscus

Similarly to articular cartilage, the ECM of meniscus is mainly composed of water (60-75% of meniscus wet weight) [6, 7, 80]. In the solid matrix, collagens are most abundant (15-25% of wet weight), while PGs account only for < 1-2% of meniscus wet weight [2, 6, 7].

About 98% of collagens in the meniscus are of type I, and the re- mainder consists of types II, III and V [2, 81]. The distribution of different collagen types shows substantial regional variation. Colla- gen type II can be found in the inner third, where it is the predominant type, as well as in the deeper superficial zone (i.e. lamellar layer) [82, 83]. In the peripheral two-thirds, the type I collagen accounts for almost all of the collagen [82]. Regional variations in the distribution of the PGs have also been observed, with the inner two-thirds having relatively higher concentration than the outer one-third [35, 84]. The ECM of the meniscus includes also small amounts of other NCPs, such as fibronectin and elastin. They help, for example, in tissue repair, blood clotting and cell migration [80].

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Knee joint

The meniscal ultrastructure consists of three distinct layers: surface layer, lamellar layer, and central main layer (Figure 2.3). The surface layer is approximately 10 µm thick and composed of a fine mesh of randomly oriented collagen fibrils [85]. In the lamellar layer, the fibrils are arranged in tight lamellar bundles and are mainly oriented radially [85, 86]. There are also vertical fibres that, assumably, tie the lamellar and central layers together allowing force transmission between the layers [85, 86]. The thickness of this layer is approximately 150–200 µm and it decreases toward the inner circumference [85].

The central layer, surrounded by the lamellar layer, forms the majority of the meniscus. The fibril bundles are predominately oriented circumferentially with few radial fibrils tying the large circumferential bundles together [2, 86, 87]. In addition, at the mid- level of the meniscus, loose connective tissue from the joint capsule penetrates radially the circumferential bundles in the external circumference [85]. The circumferential bundles are continuous with the anterior and posterior insertional ligaments [2, 78].

Figure 2.3: Superior view of the knee joint menisci (left) and illustration of the three distinct layers of the meniscus (right). In the surface layer, the collagen fibrils are randomly oriented. In the lamellar layer, the fibrils are arranged in tight lamellar bundles and are mainly oriented radially. In the central layer, which forms the majority of the meniscus, the fibril bundles are predominately oriented circumferentially. Loose connective tissue from the join capsule penetrates the meniscus radially at its mid-level.

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At birth, the whole meniscus is vascularized but only the peripheral 10-30% of adult menisci have blood vessels [76, 88, 89].

Based on the vascularity, meniscus can be cross-sectionally divided into three zones: red-red, red-white and white-white [90]. Unlike cartilage, the meniscus also has nerves in its outer two thirds; the outer third of the body is more densely innervated than the middle third [80, 91].

Meniscus contains a variety of cells. The main cell types in the inner and middle part (i.e. white-white to white-red zone) of the meniscus display features of chondrocytes and fibroblasts, and thus these cells are called fibrochondrocytes [92]. The surface layer is populated by a large number of oval or fusiform cells aligned in parallel to the articulating surfaces resembling the chondrocytes of articular cartilage [5,9,93,94]. Deeper in the main body, the cells are rounded or polygonal in shape, usually solitary, but they can also form groups of two or three [78, 92, 93]. The outer third is mainly populated by fibroblast-like cells [5].

2.2.3 Mechanical properties of meniscus

Meniscus, similar to articular cartilage, has viscoelastic properties.

Due to its highly anisotropic structure, the mechanical properties are site and orientation dependent. The shape of the menisci increases the congruency in the knee joint, which helps to distribute the loads. Indeed, the menisci play an important role in bearing and distributing loads and transmit approximately 50% of the load in the tibofemoral joint [79, 95, 96]. During axial loading, menisci are extruded tensioning the circumferential fibres and insertional ligaments [78]. Thus, part of the axial load is transformed into tensile circumferential or "hoop" stresses [9, 97]. As the collagen fibres are predominantly oriented circumferentially, menisci have great tensile stiffness in the circumferential orientation. Moreover, the circumferential tensile strength has been reported to be about tenfold to that in radial direction [2, 98–100].

Meniscus is less stiff in compression than in tension [79]. It is also about 50% less stiff and 80% less permeable than articular

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Knee joint

cartilage [10]. The low compressive stiffness and low permeability makes meniscus a good shock absorber. Under an impact, the shock is mainly absorbed by high frictional drag forces developed as the interstitial water tries to flow out of the tissue [79]. The shock absorbing capability has been shown to decrease by 20% in knees without menisci [101]. In addition, the low stiffness allows meniscus to deform, thereby conforming to the variable geometry of femoral condyles, as well as distributing the load to larger area.

2.3 CARTILAGE AND MENISCUS PATHOLOGIES

Knee joints are confronted with heavy loads and impact forces during daily life; normal walking, for example, subjects the knee joint to a load equivalent to three times the body weight [102]. Besides trauma-based injuries, articular cartilage and meniscus are suscep- tible to degenerative diseases such as OA. OA is the most prevalent disease in synovial joints, usually characterized by a progressive degeneration of the articular cartilage as well as remodeling and sclerosis of subchondral bone. Risk factors contributing to cartilage degeneration and OA include obesity, genetics, abnormal joint loading, congenital anomalies, and ageing [4, 13–15]. Cartilage degeneration results in pain, stiffness and reduced mobility of the joints. As well as decreasing the quality of life, OA is a major eco- nomical burden; in Finland, the annual cost to society due to OA has been estimated to be one billion euros [11].

Meniscal tears are one of the most common types of knee injuries, and they can be classified as acute or degenerative [103–105].

Acute tears usually occur in younger active individuals and are caused when an excessive force is applied to a healthy knee [106].

Degenerative tears are more common with older individuals, and, when a repetitive force is applied, results in a worn meniscus [90, 105]. Meniscal tears can also be categorized into multiple different types based on the pattern and location. Acute tears are often parallel to the circumferentially oriented collagen fibres (e.g. a longitudi- nal tear) but can occasionally be perpendicular to the circumferen-

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tial fibres (e.g.a radial tear) [106,107]. The latter greatly reduces the functional ability of the meniscus, and thus usually leads to meniscal surgery. Degenerative tears generally have a more complex tear pattern, and can be classified,e.g., as horizontal cleavage, oblique, or complex [90,105]. The most common location for a meniscal tear is the posterior horn of the medial meniscus [105, 108–111].

The severity and healing capability of a meniscal tear is highly location dependent. For example, lesions closer to the vascularized area have a better chance for spontaneous healing or are better targets for successful surgical repair [90, 97]. Currently, the sur- geons aim to repair an injured meniscus instead of removing it.

Treatment options include surgical repair using sutures or rigid implants (e.g. meniscus arrow or dart), but in more severe cases partial meniscectomy or meniscal allograft transplantation may be performed [9, 77, 97]. However, it must be noted that partial and especially total meniscectomy exposes the cartilage to higher peak forces, and thus to degeneration [1, 73, 96]. Nonetheless, meniscal injuries may cause further degradation of the meniscus, and thus degeneration of articular cartilage and even development of OA [16, 79, 112].

The earliest signs of articular cartilage degeneration include loss of PGs, an increase in the water content, and fibrillation of superficial collagen matrix [4, 7, 21]. These changes increase the permeability and decrease the stiffness of cartilage exposing it to further damages. In general, an increase in the water content, degradation of the collagen network and a decrease in the PG content are also related to meniscus degeneration [6, 36, 113]. However, the PG content in meniscus has also been reported to increase due to injury [113,114]. The PG content may also transiently increase deeper in the degenerated cartilage as the chondrocytes synthesize more PGs due to changed environment [21, 115].

The pathogenesis of primary OA (i.e. "wear and tear") is not yet fully understood, while secondary OA is initiated e.g. by a joint trauma. It has been postulated that impairment in the mechanical properties of articular cartilage increases stresses on subchondral

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Knee joint

bone, leading to thickening and sclerosis of subchondral bone, and thus increased loading of cartilage [116]. It has also been speculated that stiffening of subchondral bone exposes cartilage to mechanical overload and thus initiates the cartilage degeneration [117]. Fur- thermore, cartilage degeneration and OA can be consequence of a meniscal tear in a healthy knee, but OA may also lead to a meniscal tear [106, 112].

2.4 CLINICAL DIAGNOSTICS OF ARTICULAR CARTILAGE AND MENISCUS PATHOLOGIES

OA diagnosis is usually based on patient symptoms and physical examination. Common cartilage and meniscus related symptoms include pain, stiffness, effusion, catching or locking sensation, and clicking on moving the knee. To ensure the diagnosis, native X- ray image is usually taken. The severity of OA is usually assessed from the a native X-ray images by Kellgren-Lawrence grading system [118]. However, as the synovial fluid and soft tissue structures within the knee joint attenuate the X-rays almost equally, cartilage and meniscus can not be differentiated on plain radiographs. Thus, the Kellgren-Lawrence grading system is based on assessing joint space narrowing and the structural changes in bone, features that are related to moderate- to late-stage OA changes. Furthermore, cartilage and meniscus injuries can also be asymptomatic, which again delays the diagnosis of these injuries [108, 119, 120].

MRI enables detection of cartilage and meniscal lesions and evaluation of cartilage thickness [105, 121, 122]. It provides a good soft tissue contrast, and thus is the preferred imaging modality for evaluating cartilage and meniscus [17, 106]. There are several grading systems for the semi-quantitative assessment of lesions and overall knee joint health, including such as Internal Cartilage Repair Society (ICRS) grading system, Whole Organ MRI Score (WORMS) method, and Boston-Leeds Osteoarthritis Knee Score (BLOKS) method. In addition, contrast enhanced MRI techniques, such as dGEM- RIC, enable quantitative assessment of cartilage health. In this

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technique, mobile anionic gadopentate molecules are assumed to diffuse and distribute in cartilage in inverse proportion to GAGs [23, 26]. Although it is assumed that only FCD in the tissue and the charge of the contrast agent molecule affect the diffusion, it has been demonstrated that dGEMRIC is also sensitive to other factors, such as, collagen content [123–125]. This technique has also been used for imaging of the meniscus and assessment of its health [33, 36, 126]. Even though MRI provides a good soft tissue contrast, it lacks rapid 3D image acquisition with sufficient resolution. In addition, relatively high imaging costs, long acquisition and queue times limit its use in the detection of early degeneration or acute injuries.

CT arthrography (CTa) employs iodinated contrast agents to en- hance the contrast between the synovial fluid and the articular cartilage and meniscus. In CTa, the image acquisition is performed rapidly after intra-articular injection of the contrast agent. This enables morphological evaluation of cartilage and meniscus but not assessment of internal structures. CTa may be used for the detection of articular cartilage and meniscal lesions [127].

Arthroscopy is the gold standard in the evaluation of cartilage and meniscal lesions [106, 120, 128]. The modality enables direct visual inspection and probing of cartilage and meniscal surfaces, and relies on subjective assessment by the surgeon. Arthroscopic ultrasound imaging and arthroscopical optical coherence tomography may also be performed during a conventional arhtroscopic investigation. Even though these modalities allow high resolution imaging [129–132], they are not yet in widespread clinical use.

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3 Contrast enhanced com- puted tomography

The German physicist, Wilhelm Röntgen, discovered X-rays in 1895, and the use of X-rays in medical imaging was widespread already before the beginning of the 20^th century. In 1917, Johann Radon introduced the mathematical theory (i.e. Radon transform) behind computed tomography reconstruction. However, it was not until 1971 when Godfrey Hounsfield introduced X-ray computed tomography into medical practice. In CT, X-ray projections are acquired at multiple projection angles around the imaged object. Based on these projections, a 3D image of the object can be reconstructed.

3.1 X-RAY COMPUTED TOMOGRAPHY

X-rays are electromagnetic radiation with the usual wavelength of 0.01 to 100 nanometers. The X-ray imaging is based on attenuation differences between tissues. Due to various interactions with the medium, X-rays attenuate according to the Beer-Lambert law:

I(x) = I₀e⁻^µx, (3.1) where Iis the intensity of the radiation at distancex, I₀is the initial intensity, andµis the linear attenuation coefficient, which depends on the energy of the photon, atomic number (Z), and the electron density of the material. When X-ray energies are in the diagnostic range (< 200 keV), the attenuation is due to three interactions: photoelectric effect, Compton effect, and elastic scattering. The photoelectric effect is the predominant form of the interaction with diagnostic X-ray energies. In general, the photoelectric effect contributes to the image contrast whereas Compton effect contributes to image noise. [133, 134]

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In a CT scan, the X-ray source and the detector rotate around the imaged object while acquiring multiple images at multiple projection angles. These projection images cover 180 to 360 degrees of the imaged object. Subsequently, the acquired projection images are mathematically reconstructed into two-dimensional (2D) axial images using either analytical (e.g. filtered back projection) or more computationally demanding iterative reconstruction algo- rithms. Subsequently, these 2D axial images are used to form a 3D image of the imaged object. [133, 135]

Modern clinical CT scanners employ either a fan-shaped or a cone-shaped X-ray beam. The fan beam shape is achieved by extensive collimation of the X-ray beam. This reduces the amount of scattered photons reaching the detector, and thus increases the signal-to-noise ratio (SNR). Fan-beam shape is commonly applied in full-body CT scanners, with multiple detector rows (up to 320 rows). In cone beam scanners, the collimation of the X-ray beam is wider, which increases the scattering. This, with other CBCT characteristics (e.g., more complex reconstruction) results in lower SNR and contrast resolution compared with that of a fan beam scanner.

Cone beam CT (CBCT) scanners, however, typically have smaller radiation doses than fan beam CT scanners [136, 137]. CBCT scanners are commonly used in dental and extremity imaging, as well as in X-ray microtomography (microCT). MicroCT scanners are not in clinical use, but in the laboratory use they can be employed to achieve isotropic voxel size down to one cubic micrometer. To ob- tain maximal resolution with microCT, the rotating object is placed closer to the X-ray source. This magnifies the image on the detector as follows

M= ^O

0

O = ^d^so

d_sd, (3.2)

whereO⁰ is the magnified object size, Ois the original object size, d_so is the source-to-object distance, andd_sdis the source-to-detector distance.

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Contrast enhanced computed tomography

3.2 CONTRAST AGENTS

Even though soft tissues absorb X-rays, the interface between adja- cent structures (e.g., articular cartilage, meniscus and synovial fluid) can be obscured due to small differences in X-ray attenuation. Thus, contrast agents are often used to help to visualize the tissue of interest. In addition, they may be used for probing the biochemical composition or predicting biomechanical properties of the tissue.

Introduced over half a century ago [138], contrast agents are usually iodine based compounds, which can be injected intrave- nously or directly into the location of interest,e.g., intra-articularly into a knee joint. They may be non-ionic or ionic, bearing a single or multiple charges. Traditionally, the contrast agents used with X-ray CT are anionic. Recently, the use of cationic contrast agents for the imaging of articular cartilage and the meniscus was also proposed [35, 37, 38].

Generally, contrast agents should increase the attenuation differ- ence between the tissue of interest and the surrounding structures by 200% [139]. Thus, the contrast agents should contain a high atomic number. While the atomic number of iodine is relatively high (Z= 53), it is not as high as that of gold (Z = 79) or bismuth (Z = 83), which are also used as contrast agents [140, 141]. How- ever, the K-edge of iodine (33.2 keV), i.e., a sudden sharp increase in the attenuation of X-rays at the energy level corresponding to the binding energy of K-shell electrons, is almost ideal for clinically used X-ray spectra. In addition, the clinically used contrast agents should be non-toxic and cleared from the body within a reasonable amount of time (< 24 h) [139]. Contrast agents can also be tissue specific when attached to functional carrier molecules [142, 143].

3.3 DIFFUSION OF CONTRAST AGENTS IN CARTILAGE AND MENISCUS

Diffusion is a random Brownian movement of molecules or atoms from a higher concentration to a lower concentration. The state

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where the concentrations of the diffusing substance become equal, i.e., the concentration gradient disappears, is defined as the diffusion equilibrium. Diffusion and partitioning of contrast agents in cartilage and meniscus are affected by many factors. These factors will be discussed later in this chapter, however, the basic physical theory governing the diffusion and equilibration of charged particles is reviewed. The basic physical laws governing the diffusion of charged particles across a semi-permeable membrane are presented in Table 3.1.

Table 3.1: The physical laws and quantities governing diffusion of charged particles across a semi-permeable membrane.

Physical law/quantity Equation

Fick I J=−D^∂C_∂x (3.3)

Fick II ^∂C_∂t =D^∂²^C

∂²x² (3.4)

Nernst equation ∆Ψ=−^RT_zFln^[C]_[C]¹

2 (3.5)

Donnan ratio r=^[C]¹

[C]₂

1/z

(3.6)

Membrane potential ∆Ψ=−^RT_F lnr (3.7)

The opposing sides of the semi-permeable membrane are denoted with subscripts 1 and 2.

J= diffusion flux,D= diffusion coefficient,C= solute concentration,x= position,t= time,Ψ= membrane potential,R= gas constant,T= temperature (K),z= valence of the ion,F= Faraday constant,[C]= ion concentration,r= Donnan ratio.

Fick’s first law of diffusion (eq. (3.3), Table 3.1) describes diffusion in a steady state system, i.e. there is no time dependency.

Thus, the diffusion fluxJ is only related to the diffusion coefficient D and solute concentrationC. The diffusion coefficient represents the magnitude of the molar flux through a surface per unit concentration gradient, and thus describes the rate at which diffusion will reach equilibrium. It is dependent on the properties of the diffusing molecule and the medium. In the case of a time varying diffusion flux, diffusion can be described with Fick’s second law (eq. (3.4), Table 3.1). The diffusion flux of a solute across the surface of carti-

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lage or meniscus can be defined as follows J = −h∂C¯

∂t , (3.8)

wherehis the thickness of the tissue and ¯Cis the bulk concentration of the solute within the tissue [25].

The equilibrium state between two charged particles separated by a semi-permeable membrane is described by the Donnan equilibrium [144]. These charged particles (ions) create an electrical potential across the membrane. Small ions are allowed to pass through the semi-permeable membrane, but the transit of the larger ions is blocked. The smaller mobile ions diffuse across the membrane until equilibrium is reached for both electrical charge and concentration.

The Nernst equation (eq. (3.5), Table 3.1) describes the distribution of ions at equilibrium, and can be reorganized into the Donnan ratio (eq. (3.6), Table 3.1). This allows the expression of the membrane potential as presented in equation (3.7) (Table 3.1). Furthermore, the Donnan ratio can be presented as

r=1+^C¹

C₂, (3.9)

whereC₁andC₂are the concentrations of the ions on sides 1 and 2 of the membrane [144].

Let us consider a situation where tissue (cartilage or meniscus) sample is immersed and equilibrated in an electrolyte solution.

Furthermore, let us assume that mobile ions in a solute bath will distribute according to the Donnan equilibrium. When the charge of the ions is taken into account in ideal Donnan conditions, the electrochemical equilibrium in cartilage or meniscal tissue requires that [60, 145]:

[anion]_tissue [anion]_bath

zanion

=

[cation]_bath [cation]_tissue

z_cation

, (3.10) where z is the valence of the ion. Furthermore, the tissues are assumed to be externally electroneutral, and thus the following must be met:

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z_cation[cation]_bath =z_anion[anion]_bath (3.11) and

z_cation[cation]_tissue =z_anion[anion]_tissue+_{FCD ,} _(3.12) where FCD is the fixed charge density induced by the negatively charged GAGs in cartilage and meniscus [60, 146].

The equation (3.12) shows how the FCD of cartilage and meniscus affects the partitioning of charged contrast agents,i.e., anionic contrast agent molecules are electrostatically repelled by the negative fixed charge of the tissue, whereas cationic contrast agents are electrostatically attracted. This has also been proven by previous studies with anionic and cationic contrast agents [28, 35, 39, 147].

The diffusion is also affected by the organization and orientation of the collagen network, and thereby the structural integrity of the collagen matrix [25, 30, 148]. Furthermore, a previous study reported a significantly slower diffusion through deep cartilage than through the articulating surface due to the greater steric hindrance in the deep cartilage [29]. The molecular size of the contrast agent has also been shown to affect the diffusion; larger molecules are hin- dered more by the cartilage matrix [25, 29, 149]. In addition, variation in the water content relates to the porosity of the tissue, which again affects the diffusion [60].

3.4 CONTRAST ENHANCED COMPUTED TOMOGRAPHY Contrast enhanced computed tomography (CECT) is an X-ray CT technique that utilizes contrast agents to visualize and quantify the differences in tissue structures and composition. In contrast to CT arthrography, CECT is a dynamic imaging technique where multiple images are acquired at different time points. The later time points allow the evaluation of diffusion and partitioning of the contrast agent, and thus the assessment of the structure or composition of the tissue [42]. CECT usually refers to contrast enhanced techniques used for in vitro, in situ, and ex vivo measure-

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ments while in clinical in vivo measurements, the technique is referred to as delayed CT arthrography or delayed quantitative CT arthrography [150, 151]. Nonetheless, the basic principles are the same in all of these techniques.

The anionic contrast agents are assumed to distribute inversely to the FCD in cartilage or meniscus at equilibrium. With cationic contrast agents, the distribution is assumed to be directly proportional to the FCD. Indeed, previous in vitro studies have reported a strong relationship between the contrast agent concentration and PGs in cartilage and meniscus [24, 27, 35, 41, 42, 147, 152]. However, the distribution of the contrast agent is also affected by the water and collagen contents, as well as the structural integrity of the tissue matrix [29–32]. Thus, the technique might be sensitive also to other structural properties. Moreover, previous studies have shown its potential to assess the health and mechanical properties of articular cartilage [41, 153, 154].

The equilibrium time is highly dependent on the tissue thickness and the molecular size of the contrast agent. Previous studies have reported equilibrium times of over 8 hours in bovine and human cartilage with common clinical contrast agent (ioxaglate) [28, 29, 155]. Thus, reaching equilibrium is not feasible in clinical applications. However, the relationship between the contrast agent concentration and the PG concentration has been reported to be significant at an early stage of diffusionin vitro [155]. Furthermore, a recent in vivo study suggests that CECT enables a feasible tool for diagnostics and assessment of cartilage lesion already at 45 minutes after the contrast agent administration [156].

Although CECT has been widely applied to study articular cartilage in vitro [24, 25, 27, 28, 32, 40–42, 157–159], the potential of the technique to quantitatively image meniscus has not been thoroughly studied. Furthermore, the differences between the diffusion kinematics of cartilage and meniscus are still unknown.

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4 Aims of the present study

This thesis focuses on the assessment of differences in contrast agent diffusion in cartilage and meniscus.

The specific aims of this thesis were:

1. to evaluate the potential of CECT to image contrast agent diffusion in human meniscus with a clinical CBCT scanner.

2. to determine the diffusion kinematics of a small charged molecule (iodine) in cartilage and meniscus.

3. to determine the diffusion kinematics of a novel iodinated cationic contrast agent (CA2+) in cartilage and meniscus.

4. to investigate whether the diffusion of the anionic contrast agent, as imaged with CBCT in an intact human knee jointex vivo, reflects the biochemical, histological and biomechanical characteristics of articular cartilage.

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5 Materials and methods

This study comprises four independent studiesI-IV, and the main methods are summarized in this chapter (Table 5.1). For complete and detailed descriptions of materials and methods, please seeorig- inal publications.

Table 5.1: Summary of the materials and methods used in the studiesI-IV.

Study Material Methods

I human meniscus, n = 3 per meniscus, 26 menisci

CECT, MRI, biomechanics, biochemical and histological analysis

II bovine cartilage and meniscus, n= 10 for both tissues

CECT, biochemical and histological analysis

III bovine cartilage and meniscus, n= 10 for both tissues

CECT, biochemical and histological analysis

IV human cartilage,n= 17 sites CECT, biomechanics, biochemical and histological analysis n= number of samples, CECT = contrast enhanced computed tomography, MRI = magnetic resonance imaging.

5.1 SAMPLE PREPARATION

In study I, a total of 26 menisci were acquired from the left knee joints of human cadavers (n = 13, mean age 53.5 years) with permission from National Agency of Medicolegal Affairs, Helsinki, Finland (1781/32/200/01). The intact menisci were stored at -25

◦C until the experiment.

In studiesIIandIIIthe sample preparation was identical. The intact knee joints (n = 10, one per animal) were acquired from a local abattoir (HK Ruokatalo Oy, Outokumpu, Finland) within 24 hours of slaughter. The patellae and medial menisci were detached.

Subsequently, cylindrical osteochondral plugs (d = 6.0 mm) were detached from the upper lateral quadrant of the patellae and central

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region of the medial meniscus (d= 6.0 mm) (Figure 5.1). The meniscus sample was trimmed to be approximately 2.0 mm in height.

Study IV involved the right knee joint of a randomly selected cadaver (female, 91 years), obtained 4 days after death, with the permission from the ethical committee of Kuopio University Hos- pital (58//2013). The cadaver was stored at 4^◦C in the morgue at Kuopio University Hospital before the start of the experiment.

5.2 CONTRAST ENHANCED COMPUTED TOMOGRAPHY CECT images were acquired using two different scanners: a clinical CBCT scanner (studiesIandIV) and a microCT scanner (studiesII andIII). A summary of different contrast agents and CT scanners used in this thesis is presented in Table 5.2.

Table 5.2: Summary of contrast agents and different CT scanners used in studiesI-IV.

Study Contrast agent C (mgI/ml)

M (g/mol)

Voxel size (µm³) Scanner

I ioxaglate (q= -1) 48 1269 200×200×200 CBCT

II iodide (q= -1) 20 127 25×25×25 microCT

III CA2+ (q= +2) 20 686 25×25×25 microCT

IV ioxaglate (q= -1) 36 1269 200×200×200 CBCT C= contrast agent bath concentration in mg of iodine per ml,M= molar mass,q= charge of the molecule, CA2+ = contrast agent bearing two positive charges, CBCT = cone beam computed tomography, microCT = X-ray microtomography.

5.2.1 Cone beam computed tomography

A clinical CBCT scanner (Verity, Planmed, Finland) was used in studiesIandIV. A tube voltage of 96 kV, isotropic voxel size of 200

× 200 × 200 µm³ and anionic ioxaglate (q = -1, M = 1269 g/mol, Hexabrix, Mallinckrodt Inc., St. Louis, MO, USA) contrast agent were used in both studies. The contrast agents were diluted to iso- tonic concentrations of 48 mgI/ml (studyI) and 36 mgI/ml (study IV) with PBS including protease inhibitors (EDTA and benzamidine hydrochloride hydrate).

Computed tomography of cartilage and meniscus using anionic and cationic contrast agents

Natural Sciences

JUUSO HONKANEN

Computed Tomography of Cartilage and Meniscus

Using Anionic and

Cationic Contrast Agents

Preface

Contents

1 Introduction

2 Knee joint

3 Contrast enhanced com- puted tomography

4 Aims of the present study

5 Materials and methods