Martha L. Gray

Nondestructive imaging of human cartilage glycosaminoglycan concentration by MRI

Despite the compelling need mandated by the prevalence and morbidity of degenerative cartilage diseases, it is extremely difficult to study disease progression and therapeutic efficacy, either in vitro or in vivo (clinically). This is partly because no techniques have been available for nondestructively visualizing the distribution of functionally important macromolecules in living cartilage. Here we describe and validate a technique to image the glycosaminoglycan concentration ([GAG]) of human cartilage nondestructively by magnetic resonance imaging (MRI). The technique is based on the premise that the negatively charged contrast agent gadolinium diethylene triamine pentaacetic acid (Gd(DTPA)2‐) will distribute in cartilage in inverse relation to the negatively charged GAG concentration. Nuclear magnetic resonance spectroscopy studies of cartilage explants demonstrated that there was an approximately linear relationship between T1 (in the presence of Gd(DTPA)2‐) and [GAG] over a large range of [GAG]. Furthermore, there was a strong agreement between the [GAG] calculated from [Gd(DTPA)2‐] and the actual [GAG] determined from the validated methods of calculations from [Na+] and the biochemical DMMB assay. Spatial distributions of GAG were easily observed in T1‐weighted and T1‐calculated MRI studies of intact human joints, with good histological correlation. Furthermore, in vivo clinical images of T1 in the presence of Gd(DTPA)2‐ (i.e., GAG distribution) correlated well with the validated ex vivo results after total knee replacement surgery, showing that it is feasible to monitor GAG distribution in vivo. This approach gives us the opportunity to image directly the concentration of GAG, a major and critically important macromolecule in human cartilage. Magn Reson Med 41:857–865, 1999.

Protocol issues for delayed Gd(DTPA)2–‐enhanced MRI (dGEMRIC) for clinical evaluation of articular cartilage

Biochemical and histologic data have validated the technique of delayed gadolinium‐enhanced MRI, in which the T1 values of cartilage after penetration of Gd(DTPA)2–allow assessment of the glycosaminoglycan (GAG) component of articular cartilage. This work describes the factors that have been found to be important for the practical implementation of the technique: 1) Exercise immediately after intravenous contrast administration was necessary for effective penetration of the contrast into the articular cartilage; 2) double‐dose contrast was better than single‐dose; 3) after contrast administration, a time window of 30–90 min for the hip, and 2–3 hr for all compartments of the knee proved to be appropriate for assessing articular cartilage; and 4) in some cases of hypointensities in the subchondral patellar bone, decreased penetration of the contrast agent into cartilage from bone was found. With the protocol described, ROIs on T1 images were reproducible within 15% on two separate imaging sessions, and initial clinical studies demonstrated the possible applications of the technique. Magn Reson Med 45:36–41, 2001.

Assessment of Early Osteoarthritis in Hip Dysplasia with Delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage

BACKGROUND The efficacy of surgical and medical treatment of osteoarthritis is difficult to assess because of the lack of a noninvasive, sensitive measure of cartilage integrity. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) was designed to specifically examine glycosaminoglycan changes in articular cartilage that occur during the development of osteoarthritis. Our primary goal was to compare this technique with measurement of the joint space width on conventional radiographs in patients with hip dysplasia. We performed this comparison by assessing the correlation between the findings of each technique and clinically important factors such as pain, severity of dysplasia, and age. METHODS Sixty-eight hips in forty-three patients were included in the study. Clinical symptoms were assessed with use of the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) questionnaire. The width of the joint space as well as the lateral center-edge angle of Wiberg (as a measure of the severity of the dysplasia) was measured on standard standing radiographs. Magnetic resonance imaging maps of glycosaminoglycan distribution were made with T1-calculated images after administration of gadopentetate (2-) (Gd-DTPA (2-) ). The dGEMRIC index was calculated as the average of the T1 values for the acetabular and femoral head cartilages. RESULTS The dGEMRIC index correlated with both pain (rs = -0.50, p < 0.0001) and the lateral center-edge angle (rs = 0.52, p < 0.0001), whereas the joint space width did not correlate with either, with the numbers available. There was a correlation between the dGEMRIC index and pain whether or not a labral tear was present. The dGEMRIC index was significantly different (p < 0.0001) among three groups of hips classified according to whether they had mild, moderate, or severe dysplasia, whereas the joint space width did not differ significantly among these three groups. There was no significant correlation between age and any of the other parameters. CONCLUSIONS We demonstrated that, in patients with hip dysplasia, the dGEMRIC index-a measure of the biochemical integrity of cartilage-correlates with pain and the severity of the dysplasia and is significantly different among groups of hips with mild, moderate, and severe dysplasia, suggesting that it may be a sensitive measure of early osteoarthritis. Additional studies are needed to determine whether dGEMRIC can be used to predict disease progression in different situations and/or demonstrate responses to therapeutic interventions.

T2 and T1ρ MRI in articular cartilage systems

T2 and T1ρ have potential to nondestructively detect cartilage degeneration. However, reports in the literature regarding their diagnostic interpretation are conflicting. In this study, T2 and T1ρ were measured at 8.5 T in several systems: 1) Molecular suspensions of collagen and GAG (pure concentration effects): T2 and T1ρ demonstrated an exponential decrease with increasing [collagen] and [GAG], with [collagen] dominating. T2 varied from 90 to 35 ms and T1ρ from 125 to 55 ms in the range of 15–20% [collagen], indicating that hydration may be a more important contributor to these parameters than previously appreciated. 2) Macromolecules in an unoriented matrix (young bovine cartilage): In collagen matrices (trypsinized cartilage) T2 and T1ρ values were consistent with the expected [collagen], suggesting that the matrix per se does not dominate relaxation effects. Collagen/GAG matrices (native cartilage) had 13% lower T2 and 17% lower T1ρ than collagen matrices, consistent with their higher macromolecular concentration. Complex matrix degradation (interleukin‐1 treatment) showed lower T2 and unchanged T1ρ relative to native tissue, consistent with competing effects of concentration and molecular‐level changes. In addition, the heterogeneous GAG profile in these samples was not reflected in T2 or T1ρ. 3) Macromolecules in an oriented matrix (mature human tissue): An oriented collagen matrix (GAG‐depleted human cartilage) showed T2 and T1ρ variation with depth consistent with 16–21% [collagen] and/or fibril orientation (magic angle effects) seen on polarized light microscopy, suggesting that both hydration and structure comprise important factors. In other human cartilage regions, T2 and T1ρ abnormalities were observed unrelated to GAG or collagen orientation differences, demonstrating that hydration and/or molecular‐level changes are important. Overall, these studies illustrate that T2 and T1ρ are sensitive to biologically meaningful changes in cartilage. However, contrary to some previous reports, they are not specific to any one inherent tissue parameter. Magn Reson Med 51:503–509, 2004.

MRI techniques in early stages of cartilage disease.

Burstein D, Bashir A, Gray ML. MRI techniques in early stages of cartilage disease. Invest Radiol 2000;35:622–638. ABSTRACT.Cartilage degenerative diseases affect millions of people. Our understanding of these diseases and our ability to establish efficacious treatment strategies have been confounded by the difficulty of nondestructively evaluating the state of cartilage. Imaging strategies that allow visualization of cartilage integrity would revolutionize the field by allowing us to visualize early stages of degeneration and thus to evaluate predisposing factors for cartilage disease and changes resulting from interventions (eg, therapies) in culture studies, tissue-engineered systems, animal models, and in vivo in humans. Here we briefly review current state-of-the-art MRI strategies relevant to understanding and following treatment in early cartilage degeneration. We review MRI as applied to the assessment of the whole joint, of cartilage as a whole (as an organ), of cartilage tissue, and of cartilage molecular composition and structure. Each of these levels is amenable to assessment by MRI and offers different information that, in the long run, will serve as an important element of cartilage imaging.

Signal Transduction of Mechanical Stimuli Is Dependent on Microfilament Integrity: Identification of Osteopontin as a Mechanically Induced Gene in Osteoblasts

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2‐h period, and osteopontin (OPN), an Arg‐Gly‐Asp (RGD)‐containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4‐fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre‐existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA‐like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12‐myristate 13‐acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin‐D blocked the up‐regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its activity.

Magnetic Resonance Imaging of Articular Cartilage

Magnetic resonance imaging is the optimal modality for assessing articular cartilage because of superior soft tissue contrast, direct visualization of articular cartilage, and multiplanar capability. Despite these advantages, there has been disagreement as to the efficacy of magnetic resonance imaging of articular cartilage. The reason for this controversy is multifactorial but in part is attributable to the lack of the use of optimized pulse sequences for articular cartilage. The current authors will review the current state of the art of magnetic resonance imaging of articular cartilage and cartilage repair procedures, discuss future new directions in imaging strategies and methods being developed to measure cartilage thickness and volume measurements, and propose a magnetic resonance imaging protocol to evaluate cartilage that is achievable on most magnetic resonance scanners, vendor independent, practical (time and cost efficient), and accepted and used by a majority of musculoskeletal radiologists.

Magnetic resonance imaging of relative glycosaminoglycan distribution in patients with autologous chondrocyte transplants.

Gillis A, Bashir A, McKeon B, et al. Magnetic resonance imaging of relative glycosaminoglycan distribution in patients with autologous chondrocyte transplants. Invest Radiol 2001;36:743–748. rationale and objectives. Autologous chondrocyte transplantation (ACT) is a potential treatment for full-thickness chondral lesions in the knee. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) has recently been developed as a sensitive and specific measure of cartilage glycosaminoglycans (GAGs). Under the conditions of dGEMRIC, T1 is directly related to the GAG concentration. Our aim for this study was to demonstrate the potential of dGEMRIC to evaluate ACT implants. methods. Eleven ACT implants were studied 2 to 24 months postoperatively by dGEMRIC. T1 values from three regions of interest were obtained to examine GAG content (1) in the implant, (2) in native cartilage adjacent to the implant, and (3) in native cartilage further removed from the implant (as “control”). results. One implant failed and therefore was not included. Four of the implants were studied between 2 and 6 months postoperatively and showed low T1 (GAG), less than 80% of the control native cartilage. Five of the six implants studied between 12 and 24 months postoperativley showed T1 (GAG) comparable to (>80%) of control. One 18-month graft showed low T1 comparable to the surrounding native cartilage, with normal GAG seen in cartilage far from the graft site. The GAG index (T1 values of the graft normalized to control) from the group of implants 6 months or less was 59% ± 5% of control, whereas those at 12 to 24 months were 91% ± 18% of control. The two groups were statistically different with a P value of 0.005. conclusions. The GAG level in grafts that were implanted for less than 12 months appeared to be lower than that in the remote cartilage. At 12 months or greater, the grafts in this study had GAG levels that were comparable to both the adjacent and remote cartilage. This preliminary study of ACT implants has shown that it is feasible to apply the dGEMRIC technique in patients with ACT as a way to obtain information related to the composition of grafts. These results provide motivation and the pilot data with which to design further clinical studies.

Toward an identification of mechanical parameters initiating periosteal remodeling : a combined experimental and analytic approach

The ability of bone to adapt to its mechanical environment is well recognized, although the specific mechanical parameters initiating or maintaining the adaptive responses have yet to be identified. Recently introduced mathematical models offer the potential to aid in the identification of such parameters, although these models have not been well validated experimentally or clinically. We formulated a complementary experimental/analytic approach, using an animal model with a well-controlled mechanical environment combined with finite element modeling (FEM). We selected the functionally isolated turkey ulna, since the loading could be completely characterized and the periosteal adaptive responses subsequently monitored and quantified after four and eight weeks of loading. Known loads input into a three-dimensional, linearly elastic FEM of the ulna then permitted full-field mechanical characterization of the ulna. The FEM was validated against a normal strain-gaged turkey ulna, loaded in vivo in an identical fashion to the experimental ulnae. Twenty-four candidate mechanical parameters were then compared to the quantified adaptive responses, using statistical techniques. The data supported strain energy density, longitudinal shear stress, and tensile principal stress/strain as the mechanical parameters most likely related to the initiation of the remodeling response. Model predictions can now suggest new experiments, against which the predictions can be supported or falsified.

New MRI Techniques for Imaging Cartilage

Because of its ability to image all of the tissues in a diarthrodial joint, magnetic resonance imaging (MRI) has an ever-increasing role in the evaluation, diagnosis, and monitoring of joint disorders. Standard MRI techniques can delineate morphologic abnormalities. Techniques on the horizon offer improved morphologic analysis as well as previously unavailable information about the biochemical composition and functional properties of joint tissues. While research and development efforts are rapidly growing, the current review focuses on techniques that are most advanced and that have demonstrated feasibility in basic science and clinical studies. As such, we report mainly on cartilage imaging but hasten to add that ongoing research efforts offer promise for the imaging of all joint structures. These techniques should improve our ability to understand the healthy joint and the disease process, to provide earlier diagnoses, and to evaluate the effects of therapeutic procedures. With these capabilities, we can more effectively establish strategies to maintain joint health and to identify indications for intervention at an early stage of degeneration. We begin with an overview of the basics of MRI to provide the reader with a sense of what is measured and how an MR measurement is made. MRI is possible because nuclei with an odd number of protons and/or an odd number of neutrons have net magnetic moments. The most abundant nucleus in biological systems is the hydrogen nucleus (which, in MR vernacular, is referred to as a proton because hydrogen contains a single proton), making water and fat both observable with MRI. In all of the methods that we will discuss here (and virtually all of the MR imaging done clinically), it is the hydrogen (proton) nucleus that is being measured; indeed, the term MRI has come to be synonymous with the term proton-MRI (while MR of another nucleus x …