Visualizing Tissue Deformation by Noninvasive MRI
Alteration of the mechanical function of musculoskeletal tissue following acute injury or progression of disease can be detrimental to daily activities, such as walking. The breakdown of articular cartilage in osteoarthritis serves as an example of progressive degradation and inferior tissue mechanics (ie, reduced tissue stiffness). Restoration of tissue mechanics is one design end point for strategies of regenerative medicine and tissue engineering. To this end, the measurement of in vivo deformation (eg, displacement, strain patterns) in native and repair tissue is required to establish the patterns of activity and performance, clarify the limits of expected use, and define standards of success for tissue repair.1
Magnetic resonance imaging (MRI) is a noninvasive experimental technique with the potential to measure detailed tissue deformation in vivo. MRI can visualize morphology with excellent contrast of soft tissues, and it can characterize physical phenomena such as diffusion and flow. Significant advances in MRI techniques, or pulse sequences, permit direct measurement of motion within the tissue interior without the use of markers or instrumentation that may influence the tissue deformation being measured. Although measurement of motion has a long history in nuclear magnetic resonance,2 modern motion-sensitive techniques exploit both magnitude and phase data from the complex magnetic resonance signal. The magnitude image is the image most commonly visualized and familiar to users, such as clinicians.
Magnitude images may be used to describe surface and interior tissue motion. The measurement of surface motion is perhaps an obvious application of magnitude images. Typically, high spatial resolution images are acquired for a tissue (eg, articular cartilage) before and after an exercise or imposed mechanical load; imageprocessing techniques subsequently determine tissue boundaries and relative thickness or volume changes.3 However, such measurements do not capture the heterogeneous motion occurring within the tissue. Interior tissue motion requires texture correlation using the natural features available in images of tissue (eg, signal density variations in trabecular bone or tendon4,5) or patterns superimposed directly and noninvasively on the tissue (eg, tag lines in articular cartilage6).
A general assumption made about these techniques is that the features of interest are visualized in images of both nondeformed and deformed tissue without significant out-ofplane motion to minimize errors in the measurement. One application of texture correlation in soft orthopaedic tissues documented intratissue motion in tendons and ligaments with a subpixel displacement error (of approximately 25% of pixel spatial dimensions).5 More recent techniques include the use of tissue “tagging” to allow direct observation of motion in articular cartilage.6 Texture correlation has been used in conjunction with optical techniques7 that, although not easily applicable in vivo, provide a higher spatial resolution of deformation data compared with MRI.
Phase images also have been used to characterize intra-tissue motion. In contrast to texture correlation, in which the features of an object in magnitude images may be separated by several pixels, phase-contrast methods have the ability to directly compute the deformation occurring within each image pixel—a significant advantage. The density of data characterizing a deformation field is thus directly related to the image spatial resolution. A technique using displacement encoding with stimulated echoes (DENSE), followed by a fast spin-echo readout, has quantified tissue deformations at a spatial resolution of 100 × 100 μm2 in articular cartilage explants during physiologically relevant applied stress levels and loading rates.8 The technique quantified strain with a precision better than 0.17% and thus was appropriate to characterize the micromechanical strain environment in cartilage in response to applied mechanical loading. In vivo displacements and strains have been documented using phase-based MRI techniques in muscle.9 The pixel dimensions for this in vivo technique were 1.9 × 1.9 mm2. Although these dimensions are not appropriate for small orthopaedic tissues, they are sufficient to provide novel detail regarding mechanics of muscle tissue to a level that has not previously been possible.
A major challenge in characterizing in vivo deformation throughout orthopaedic tissues is related to the spatial scales: small tissues, such as articular cartilage, require images with high spatial resolution. Demand for small pixels lies in the need to determine or diagnose local regions of disease, repair, and regeneration.
Corey P. Neu PhD
The Role of Epigenetics in Skeletal Development and Diseases
DNA is the blueprint of life, but the genetic instructions received from our parents are now known to be modified as we develop and age. These modifications have been termed epigenetic to denote adaptive changes that occur in addition to changes in the genetic code (hence the Greek prefix epi-).
Although epigenetic changes are essential and determine cell fate and tissue differentiation very early in development, extrinsic environmental factors (eg, nutrition, stress, exposure to carcinogens) can affect postnatal genome function via epigenetic pathways.10 These extrinsic influences are strikingly demonstrated by the marked epigenetic differences that exist between adult monozygotic twins, who begin postnatal life as both epigenetically and genetically identical.11 The observation that epigenetic changes are associated with many diseases has stimulated the development of drugs targeting epigenetic regulatory enzymes. Aging-associated diseases (eg, arthritis) are prime candidates for the cumulative effects of epigenetic changes and thus are prime targets for epigenetic therapeutics.
Epigenetics refers to changes in gene expression that do not involve changes in the underlying DNA sequence itself. Epigenetic changes are inherited by daughter cells and are sometimes passed on to offspring. Epigenetic changes in gene expression can occur by modification, including acetylation and methylation, of the histone proteins responsible for DNA packaging into chromatin. Changes also can occur by direct effects on DNA, including methylation of cytosine, where it is followed by a guanine residue (CpG). Somewhat simplistically, regions of transcriptionally inactive DNA are marked by widespread CpG methylation and by deacetylated histone proteins. Conversely, transcriptionally active genes contain unmethylated CpG dinucleotides and are arranged within and around acetylated histone-DNA complexes. Decreased gene activity is associated with enzymes that catalyze DNA methylation (DNA methyltransferases) and histone deacetylation (histone deacetylases [HDACs]). Increased gene activity is associated with enzyme families the catalyze histone acetylation (histone acetyl transferases [HATs]).
Epigenetic processes are responsible for such well-characterized phenomena as X chromosome inactivation, heterochromatin silencing, and gene imprinting. In these instances, specific genes or entire chromatin stretches are transcriptionally suppressed by epigenetic alterations. Epigenetic effects are also critical to the success and failure of cloning technologies and influence the self-renewal capacity of stem cell populations.
HDACs regulate cellular differentiation in a range of cell lineages, as exemplified by recent work that addresses the role of HDAC activity in skeletal development.12 Several HDACs negatively interact with transcription factors critical for osteogenesis and antagonize osteoblastic differentiation. Consistent with these findings, pharmacologic inhibitors of HDAC activity stimulate differentiation of osteoblasts. Analogously, mice lacking the gene HDAC413 exhibit a severe phenotype reflecting unconstrained chondrocyte differentiation, with osseous replacement of normally permanent articular and costal cartilages. The HDAC4 null phenotype is linked to deregulated Runx2 activity in affected chondrocytes. Analogous to HDAC activities in bone cells, HDAC4 appears to be necessary for appropriate rates of chondrocyte differentiation and suppression of terminal differentiation in permanent cartilage tissues. Collectively, these studies suggest that key lineage-specific transcription factors in osteoblasts and chondrocytes are regulated by chromatin-modifying enzymes in both positive and negative fashions.
Aberrant epigenetic activities have been linked to several diseases, including cancers, immune-mediated diseases, and psychiatric conditions. Several lines of evidence have implicated epigenetic processes in arthritic disease. The CpG methylation profiles of several matrix metalloproteinase (MMP) genes are reduced in osteoarthritic chondrocytes, concurrent with increased expression and activity of these proteinases in arthritic cartilage.14 Furthermore, HDAC inhibitors block in vitro MMP expression and activity by human articular chondrocytes exposed to catabolic cytokines.15 Here, HDAC inhibitors may stimulate expression of a gene that codes for an MMP repressor. Both CpG methylation and histone acetylation abnormalities have been implicated in immune cell dysfunction and synovial fibroblast hyperplasia in rheumatoid and inflammatory arthritis.10,16 Consistent with these findings, several HDAC inhibitors mitigate disease severity in rodent inflammatory/rheumatoid arthritis models. In these models, HDAC inhibitors suppress the otherwise excessive host inflammatory response and reduce synoviocyte proliferation.16
Studies of the role of epigenetics in skeletal development and disease are in their infancy. We can be sure that the rapidly increasing understanding of epigenetics will have a major impact on our understanding of skeletal diseases and their treatment.
Matthew Stewart BVSc, PhD, FACVSc
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