Musculoskeletal molecular imaging: a comprehensive overview

Abstract

Molecular imaging permits non-invasive visualization and measurement of molecular and cell biology in living subjects, thereby complementing conventional anatomical imaging. Herein, we review the emerging application of molecular imaging for the study of musculoskeletal biology. Utilizing mainly bioluminescence and fluorescence techniques, molecular imaging has enabled in-vivo studies of (i) the activity of osteoblasts, osteoclasts, and hormones, (ii) the mechanisms of pathological cartilage and bone destruction, (iii) skeletal gene and cell therapy with and without biomaterial support, and (iv) the cellular processes in osteolysis and osteomyelitis. In these applications, musculoskeletal molecular imaging demonstrated feasibility for research in a myriad of musculoskeletal conditions ranging from bone fracture and arthritis to skeletal cancer. Importantly, these advances herald great potential for innovative clinical imaging in orthopedics, rheumatology, and oncology.

Figure I

The principle of molecular imaging.

Figure 1

Strategies for conferring visibility in musculoskeletal molecular imaging. Bioluminescence imaging is frequently based on the Firefly luciferase (FLuc) reporter gene. Alternative luciferases used for bioluminescence imaging are Renilla or Gaussia luciferase, which catalyze the production of CO₂ and light (Em ~480 nm) in a cofactor-independent manner from coelenterazine and its derivatives instead of D-luciferin. The bacterial lux operon provides a basis for bioluminescence imaging without the need for exogenous substrate. Other reporter genes are the green fluorescent protein (GFP) for fluorescence imaging as well as the nuclear imaging reporter gene Herpes simplex virus 1 thymidine kinase (HSV1 TK), which relies on the preferential phosphorylation and hence cellular trapping of HSV1 thymidine kinase substrates. Employing probes represent a second important strategy for conferring imaging visibility. Two different types of probes can be used: static and activatable. Static probes that have been modified with a fluorophore can be imaged following their in-vivo concentration at a target site. Based on the observation that light absorption through biological tissues decreases significantly at wavelengths longer than approximately 600 nm, reaching a minimum around 750 nm [91], near-infrared (NIR, approximately 700 –900 nm) fluorophores are preferred optical labels for fluorescence imaging. Nuclear imaging of isotope-labeled static probes is also an established method. Popular isotopes for imaging applications include the gamma-emitters technetium-99m and indium-111 or the positron emitters fluorine-18 and copper-64. In contrast to static probes, activatable probes gain fluorescence through enzyme-mediated release of active fluorophores from a quenched precursor. Musculoskeletal molecular imaging studies have utilized a probe with a relatively broad selectivity to proteinases, including cathepsin B, L, S, and plasmin [32,33] or a probe preferentially cleaved by cathepsin K [10]. Utilizing antibodies instead of probes is an established strategy and includes fluorophore-labeled antibodies for fluorescence imaging and anti bodies that carry an isotope and thus are suitable for nuclear imaging. Particles composed of a cell membrane-penetrating shell and a paramagnetic iron-oxide core are used for cell labeling and subsequent detection on conventional proton MRI. Abbreviations: Ex, excitation wavelength; Em, emission wavelength; PET, positron emission tomography; γC/ SPECT, gamma camera/single photon emission computed tomography.

Figure 2

Applications of musculoskeletal molecular imaging. (a) Two representative images of female OC-FLuc transgenic mice at 6–8 weeks of age. Bioluminescence imaging could detect osteocalcin activity in areas of active bone remodeling, including calvaria bones, teeth area, paws and proximal tail vertebra e. Images courtesy of Dr. Yoram Zilberman, Skeletal Biotech Laboratory (Professor Dan Gazit), Hebrew University-Hadassah Medical Center. (b) Imaging macrophage infiltration into inflamed joints using a NIR fluorophore-conjugated folate. The top panel shows a white-light image and the bottom panel the merged fluorescence image in which the onset of inflammation was detected 30 h after induction of arthritis in the right wrist. The color bar indicates signal intensity ranging from low (white) to high (red). Reproduced with permission from [18]. (c) Fluorescence molecular tomography of bone formation at the mineralization stage utilizing a NIR fluorophore-tagged bisphosphonate. The image shown was acquired 3 weeks after transfer of BMP-2-enhanced MSCs into a radial bone defect and showed a signal along the defect site. The shoulder of the animal is marked by an arrow. The color bar indicates signal intensity ranging from low (violet) to high (red). Reproduced with permission from [72]. (d) Temporal-spatial homing of macrophages to a bone cement particle-challenged femur was revealed by a series of bioluminescence images taken every 48 h over 10 days (from left to right). The color bar indicates signal intensity ranging from low (violet) to high (red). Reproduced with permission from [73].