Advances in Noninvasive Functional Imaging of Bone

Advances in Noninvasive Functional Imaging of Bone Sheng-Min Lan, MD, Ya-Na Wu, PhD, Ping-Ching Wu, PhD, Chi-Kuang Sun, PhD, Dar-Bin Shieh, DDS, DMSc, Ruey-Mo Lin, MD The demand for functional imaging in clinical medicine is comprehensive. Although the gold standard for the functional imaging of human bones in clinical settings is still radionuclide-based imaging modalities, nonionizing noninvasive imaging technology in small animals has greatly advanced in recent decades, especially the diffuse optical imaging to which Britton Chance made tremendous contributions. The evolution of imaging probes, instruments, and computation has facilitated exploration in the complicated biomedical research field by allowing longitudinal observation of molecular events in live cells and animals. These research-imaging tools are being used for clinical applications in various specialties, such as oncology, neuroscience, and dermatology. The Bone, a deeply located mineralized tissue, presents a challenge for noninvasive functional imaging in humans. Using nanoparticles (NP) with multiple favorable properties as bioimaging probes has provided orthopedics an opportunity to benefit from these noninvasive bone-imaging techniques. This review highlights the historical evolution of radionuclide-based imaging, computed tomography, positron emission tomography, and magnetic resonance imaging, diffuse optics–enabled in vivo technologies, vibrational spectroscopic imaging, and a greater potential for using NPs for biomedical imaging. Key Words: Bone; functional imaging; molecular imaging; diffuse optics; nanoparticles. ªAUR, 2014

THE EVOLUTION OF STRUCTURAL IMAGING TO FUNCTIONAL AND MOLECULAR IMAGING OF BONE

B

one is a composite material consisting primarily of hydroxyapatite crystal and type I collagen, which accounts for
90% of the organic component. Because bone has high mineral content and higher attenuation of xrays than the surrounding soft tissue, conventional roentgenography was, when introduced in 1895, the first noninvasive structural imaging modality for bone. Because of the advances in physics, digital geometric processing, and computational power, computed tomography (CT) and magnetic resonance imaging (MRI) can process a large array of digital data to Acad Radiol 2014; 21:281–301 From the Department of Orthopaedics National Cheng Kung University Medical Center Dou-Liou Branch, Yunlin, Taiwan (S.-M.L., R.-M.L.); Institute of Oral Medicine and Department of Stomatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University Tainan 701, Taiwan (Y.-N.W., P.-C.W., D.-B.S.); Molecular Imaging Center, Graduate Institute of Photonics and Optoelectronics, National Taiwan University, Taipei, Taiwan (C.-K.S.); Department of Electrical Engineering, National Taiwan University, Taipei, Taiwan (C.-K.S.); Center for Micro/Nano Science and Technology, National Cheng Kung University, Tainan, Taiwan (D.-B.S.); Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan, Taiwan (D.-B.S.); and Department of Orthopedics, Division of Spinal Surgery, College of Medicine, National Cheng Kung University, Tainan, Taiwan (R.-M.L.). Received October 9, 2013; accepted November 26, 2013. Supported by grants NSC 102-2120-M-006-003-MY3 and NSC 1012314-B-006-048-MY3 from the Taiwan National Science Council, and a grant from National Cheng Kung University’s Headquarters of University Advancement, which is sponsored by the Taiwan Ministry of Education. Address correspondence to: D.B.S. e-mail: [email protected] or R.-M.L. e-mail: [email protected] ªAUR, 2014 http://dx.doi.org/10.1016/j.acra.2013.11.016

reconstruct virtual three-dimensional (3D) images of bone and soft tissue (1). However, functional changes usually precede structural changes in disease process, and molecular signaling aberrations are fundamental to most diseases. Therefore, clinical demands for advanced imaging technology capable of providing functional and molecular information are urgent. From the functional point of view, physiological bone homeostasis, repair, and pathologic changes are mediated by a balance of positive and negative remodeling processes. Noninvasive functional imaging of bone includes in vivo imaging of bone formation, resorption, inflammation, vascularization, and mechanical properties. At the molecular level, the bone-remodeling kinetics involves a complex molecular interaction network between the cells and the cell–matrix. Appropriate functional and molecular imaging systems with sufficient spatial and temporal resolution provide valuable information to advance bone research and clinical frontiers. Functional imaging, compared to structural imaging, unravels physiological and pathologic activities, and it has been widely used both in basic medical research and in clinical settings. The scope of functional imaging is shown in Figure 1. Functional imaging senses signals from biological tissue and reconstructs the information into registered images that reflect regional changes in the blood supply, metabolism, chemical constituents, or physical properties. Decades ago, the introduction of intravascular contrast agents not only allowed physicians to distinguish vessels from other tubular structures in the human body but also shed a light on functional imaging (2). Contrast-enhanced CT has been used to assess vascularity 281

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its derivatives (20,22,23), alizarin red derivatives (20,23,24), and xylenol orange (20,25). Although the mainstream targeting unit for skeletal imaging is still bisphosphonate derivatives, the reporter unit has substantially advanced in the past decades. The Reporter Unit of Bone-imaging Probes: From Radionuclides to Near-infrared Fluorophores to Nanomaterials

Figure 1. The scope of noninvasive molecular, functional, and structural imaging for the skeletal system. Noninvasive functional imaging reflects changes in the blood supply, metabolism, and chemical constituents or physical properties (electrical or optical), which covers imaging at the molecular to tissue level.

and permeability to distinguish between benign and malignant lesions in orthopedics and other clinical specialties (3–7). Molecular imaging (8–10), an integral part of functional imaging, is recognized as an important medical advance toward personalized medicine to optimize disease treatment. Detecting subtle functional changes at the molecular level provides advanced diagnoses and clues to therapeutic strategies. The development of molecular probes and new sensing and image processing technologies has contributed to the emergence of molecular imaging. To map the molecular events in vivo, the imaging probes consist of a targeting unit that directs the probe to the site of interest and a reporter unit that emits signals spontaneously or on specific stimulation. The Targeting Unit of Bone-imaging Probes

Bisphosphonates or diphosphonates, used to treat osteoporosis and similar diseases, preferentially bind to bone (11). Alendronate (a class of bisphosphonates), with a dissociation constant (Kd) of about 103 mol/L at pH 7 for bone hydroxyapatite (12), is a good targeting unit for calcium-rich tissue. Ozcan et al. (13) synthesized poly(g-benzyl-l-glutamate) polypeptidederived NPs by conjugating alendronate and fluorescein isothiocyanate (FITC). After mice had been intravenously injected with such NP, they were found in the bone tissue only ex vivo but could not be noninvasively detected. A range of distinct glutamate-like receptors and their associated proteins are expressed in both osteoblasts and osteoclasts (14). L-Glutamate binds to osteoblasts (
104 KD mol/L) (14), and it has been reported (15,16) to be a targeting unit for bone tissue; however, most reports are still limited to in vitro experiments. Because glutamate-like receptors are rich in neurons (17), the practicality of using glutamate as a bone-cell–seeking ligand warrants further evaluation. Other less reported hydroxyapatite-targeting probes include phosphonate derivatives (18), divalent cation chelating agents such as calcein (19), DCAF (2,4-bis[N,N0 -di(carboxymethyl)–aminomethyl] fluorescein) (20,21), tetracycline and 282

Radionuclides, with the advantages of highly penetrative grays and being ready to integrate into or with the targeting unit, opened up a wide opportunity for functional and molecular imaging both in clinical settings and in basic research. Despite the merit of high sensitivity and deep tissue penetration, functional imaging using radionuclides harbors the drawback of potential biological hazards and high cost. Therefore, scientists started to search for alternative imaging methods to obtain information from bone. In classical optics, ballistic photons are used to form images, but scattered photons are regarded as noise. The number of ballistic photons decreases exponentially because of the scattering and absorption. The inhomogeneous biological composition of skin, fat, muscle, fibrous tissue, vessel, lymphatics, osseous tissue, and pigments such as cytochromes, hemoglobin, and melanin results in remarkable scattering and absorption of photons, which severely compromises spatially resolved or spectroscopic imaging of deep tissue-like bone. In addition, visible and ultraviolet lights produce significant autofluorescence in biological tissue, which severely compromises the signal-to-noise ratio. With the advances in photonics and algorithmic modeling of photons absorbed and scattered in turbid media, as in most biological tissue greater than a certain thickness, valuable information can now be retrieved from an array of detectors that collect a plenitude of diffuse photons from the deep photon sources to reconstruct spatially resolved images. Britton Chance made significant contributions to diffuse optical tomography (DOT) (26–31). As early as 1989, Chance et al. (29) developed a model based on the diffusion approximation to radiative transfer theory. The model predictions, highly comparable to in vivo results and Monte Carlo simulations, are a milestone for diffuse optics. In 2002, Chance et al. (30) developed the amplitude cancellation method to sensitively and accurately detect and locate small objects in turbid media. This breakthrough facilitated the practicality of using nonionizing light emitters in functional imaging in a variety of clinical settings. Near-infrared (NIR) light has garnered attention because of its biological safety and low tissue absorption coefficient (32). There are two biological windows for NIR: 650– 950 nm (NIR I) and 1000–1350 nm (NIR II) (32). The NIR I penetrates the human body very well, and selective NIR spectra are able to differentiate oxyhemoglobin from deoxyhemoglobin to reveal perfusion and oxygenation of deep target organs and to noninvasively study brain cortical

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activation (33,34). NIR I is much less scattered than visible light in biological tissue and has been widely used in smallanimal in vivo imaging. The U.S. Food and Drug Administration (FDA)–approved fluorophores excited at NIR I include indocyanine green and 5-aminolevulinic acid. NIR II has been heavily investigated in the recent years because of better biological optical properties, advances of NIR cameras, and the enhancement of NP fluorophores in this spectral domain. Scattering and background noise from tissue autofluorescence is even lower for NIR II than for NIR I (35–37). Hence, the spatial and temporal resolution is improved with an extended imaging depth (38). Several types of potential nanoprobes have been developed, such as single-walled carbon nanotubes (39), Ag2S quantum dots (40), and core-shell gold NPs (GNPs) (41). However, the experimental nanoprobes working at NIR II have not yet been FDA-approved for clinical use. The development of nanotechnology in the past few decades has extensively advanced current medicine. Nanomaterials have structural features that fall within the gamut of atoms and bulk materials. Most microscale materials have properties similar to those of the corresponding bulk materials. However, many nanoscale materials have been found to possess novel physical properties (e.g., magnetic, optical, thermal, and acoustic) and favorable surface chemistry (42,43) and are excellent candidates for reporter units and vehicle. Because nanomaterials and biomolecules are similar in size, nanomaterials are used as vehicles to deliver molecules and manipulate certain biomolecules for medical purposes (44,45). Some NPs have unique properties well suited for medical imaging, such as super paramagnetic iron oxide NPs (SPION) for MRI, and GNPs for CT. Nanomaterial-enabled unique molecular and functional imaging has been extensively studied (46,47). Some nanomaterial-based functional imaging contrast agents have been approved and used clinically (48). In the following sections, the imaging modalities used for the skeletal system, including radionuclide imaging of bone metabolic activity and DOT for bone activities and noninvasive assessment of bone quality, will be reviewed. Functional imaging modalities for bone available for clinical use are summarized in Table 1 whereas those still in research are summarized in Table 2. Finally, the potential development of nanomaterial-enabled imaging in orthopedics will be discussed. IMAGING MODALITIES FOR THE SKELETAL SYSTEM Radionuclide Imaging of Bone Metabolic Activity

Radionuclide bone scintigraphy is currently the gold standard for functional bone imaging. Its clinical applications include detecting occult fracture, benign and malignant bone tumors (including those of primary and metastatic diseases), infection, and avascular osteonecrosis. Bone-seeking radiotracers accumulate on the surface of or within the crystalline structure of hydroxyapatite after they have been intravenously injected.

The efficiency of radiolabeling the bone depends on the local blood flow that supplies bone tissue, the exposed surface area of the bone, capillary permeability, and the kinetics of radiotracer incorporation with bone. The radioactively labeled molecules then undergo a homoionic or heteroionic exchange with molecules native to bone (49). Microautoradiographic studies (50) showed that 99mTc deposition occurs at the mineralization front of bone (osteoid) and at the borders of osteocyte lacunae. There is no radiotracer detected in the cytoplasm or nucleus of any osteoblast, osteocyte, osteoclast, or Howship’s lacuna. In contrast to 99mTc, a fraction of extravascular fluoride is directly incorporated into the bone matrix to form fluoroapatite (51). The net amount of fluoride transported into the bone correlates with bone metabolic activity, which was proved by studies using bone histomorphometry (52,53), the gold standard for quantifying bone formation. Early attempts at bone-seeking radioisotopes took advantage of the homoionic exchange in which the b-particle is emitted from 32P and 45Ca. Because the b-particle has a much shorter range of even energy (
1 cm) and a higher ionizing power in biological tissue, 32P and 45Ca are replaced by heteroionic g-ray emitters, such as 87mSr (Eg = 388 keV) for Ca and 18F (Eg = 511 keV) for the hydroxyl group in hydroxyapatite. Sodium fluoride (NaF) labeled with 18F (18F-NaF), introduced in 1962, was the first widely used agent for skeletal scintigraphy (54); in 1972, the U.S. FDA approved it for clinical use. 18F-NaF has high and rapid bone uptake accompanied by very rapid blood clearance, which implies a high bone-to-background ratio in a short time. Highquality skeletal images can be obtained within 1 hour after an intravenous injection. The images had to be acquired with rectilinear scanners equipped with thick NaI(Tl) (sodium iodide doped with thallium) crystals because of the relatively high energy of the 511-keV annihilation photons produced by the decay of 18F. Then came the new thincrystal g-cameras, which were optimized for imaging with 99m Tc (Eg = 140 keV) (55). Combined with the widespread availability of 99Mo/99mTc generators, 99mTc-methylenediphosphonate and 99mTc-hydroxymethylene diphosphonate took the place of 18F-NaF bone scans in the mid 1970s. Imaging shortly after a 99mTc-diphosphonate injection assesses vascularization and perfusion–namely, the blood-pool images that correlate with increased metabolic activity (e.g., bone infection and tumor). It is necessary to wait about 3 hours after the 99mTc-diphosphonate injection before bone imaging because its significant plasma protein binding hinders clearance. Single photon emission computed tomography (SPECT) provides improved resolution of smaller regions of interest at the cost of time. Positron emission tomography (PET) scanners have higher spatial resolution and substantially greater sensitivity than do conventional g-cameras. PET/CT, combined PET and CT scanning, allows not only accurate anatomic localization of the lesion but also increased resolution because of attenuation correction parameters derived from CT. Moreover, the supply of 18F-NaF is facilitated by an efficient commercial system 283

Modality

99m

Image acquisition time Spatial resolution* Detection depth Sensitivityy

Minutes 7–15 mm Whole body 1010 to 1011 mol

Tc-MDP SPECT

18

F-NaF PET

Contrast-enhanced CT

Dual-energy CT

Minutes Minutes 0.5–2.0 mm <1 mm Whole body Whole body 1. 102 to 103 mol Greater than CT organic contrast 2. 109 to 1010 for nanoparticle Specificity Low Greater than SPECT 1. Low for pure contrast High 2. High for molecular- targeting contrast probe. Volumetric quantification Relative Absolute Absolute Absolute Cost of infrastructure High Higher than SPECT Moderate Moderate Cost of each use Moderate High Low Low 1. Gout crystal Detection applications 1. Blood flow 1. Blood flow 1. Blood flow 2. Bone marrow edema 2. Metabolism 2. Metabolism 2. Vascular 3. Ligament permeability 4. Metal artifact 3. Molecular reduction 4. Calcification Major advantages 1. High sensitivity 1. High spatial resolution 2. Whole body depth access 2. Excellent bone and implant structural imaging 3. Fast image acquisition

1. Relatively low spatial resolution 2. Ionizing radiation 3. Higher cost infrastructure

1. 2. 3. 4.

Poor soft-tissue discrimination Lower contrast sensitivity Ionizing radiation Risk of contrast agent–induced adverse effects

Doppler Ultrasound

Minutes to hours 10–100 mm Whole body 103 to 105 mol for Gd contrast agent 106 to 109 mol for selective nanoparticles High

10–30 milliseconds 50 mm Centimeters —

Absolute High Low 1. Blood flow 2. Molecular 3. Bone marrow edema

— Low Low 1. Blood flow

1. Good soft tissue and spatial resolution 2. Multiple imaging sequences 3. Whole body depth access 1. Slow image acquisition 2. Higher cost 3. Risk of contrast agent–induced adverse effects for patients with endstage renal disease

1. Real-time image acquisition 2. High spatial resolution 3. Relatively Lower cost 4. More portable 1. Operator-dependent result 2. Limited depth access 3. Quantification

—

CT, computed tomography; MDP, methylenediphosphonate; MRI, magnetic resonance imaging; NaF, sodium fluoride; PET, positron emission tomography; SPECT, single photon emission computed tomography. Data are integrated from the previous studies (9,10,79,115,218–224). *The listed spatial resolutions of SPECT, PET, and CT are those in clinical use. Those for small-animal use could reach a spatial resolution at 0.5–2 mm for microSPECT, 1–2 mm for microPET, and 20–300 mm for microCT. The spatial resolution of SPECT depends on the detectors’ intrinsic resolution, collimator design, and source-to-collimator distance; it can be greater than that of PET in some circumstances. The spatial resolution of ultrasound can be separated into axial and lateral resolution. The spatial resolution is affected by frequency, depth, and spatial pulse length. y Nanotechnology can increase the in vitro detection sensitivity of MRI/photoacoustic images upto 1012 level (217).

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Major disadvantages

Shorter than SPECT 6–10 mm Whole body 1011 to 1012 mol

Proton MRI

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TABLE 1. Current Noninvasive Functional Imaging Modalities in Orthopedic Clinics and Research

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TABLE 2. Current Noninvasive Functional Imaging Modalities in Orthopedic Research Only

Modality

Fluorescence Reflectance Imaging (FRI)*

Bioluminescence Imaging (BLI)

Energy source

Chemical luminescence

Image acquisition time Spatial resolution Detection depth Sensitivity

Fluorescence Molecular Tomography (FMT)

Photoacoustic Imaging (PAI)

Near-infrared

Near-infrared laser

Seconds to minutes 3–5 mm <3 cm 1015 to 1017 mol

1. Visible light 2. Near-infrared Seconds to minutes 1–3 mm <1 cm 109 to 1012 mol

Minutes <1 mm <20 cm 1013 mol

Specificity

High

High

High

Volumetric quantification Cost of infrastructure Cost of each use Detection capability

Relative Moderate Low Molecular

Relative Moderate Low 1. Molecular 2. Perfusion

Absolute Moderate Low 1. Molecular 2. Perfusion

Major advantages

1. Very high sensitivity 1. Deeper imaging than BLI, if NIR probe used 2. Gene expression 2. Wide options quantification of of probes intracellular proteins 3. Multiplexed imaging 3. Faster image acquisition than FMT 4. Multiplexed imaging 1. 2D imaging 1. Need genetic 2. Limited depth engineering of access 2. Superficial imaging 3. Relatively lower 3. 2D imaging spatial resolution 4. Relatively lower spatial resolution

Milliseconds
100 mm
5 cm 1. 109 mol 2. 1012 mol for nanoparticle contrast High with targeting contrast Relative Low Low 1. Molecular 2. Perfusion 3. Vasculature 4. Anatomic 1. Good spatial resolution 2. Relatively deep access 3. Real-time imaging

Major disadvantages

1. Relatively deep access 2. High 3D resolution 3. Absolute quantification

Longer image processing Relatively lower sensitivity time than BLI and FRI

NIR, near-infrared; 2D, two-dimensional; 3D, three-dimensional. Data are integrated from the previous studies (9,10,79,218,222,225). *The FRI using NIR II probes can shorten the image acquisition time to <200 milliseconds and increases the spatial resolution to about 30 mm (37).

driven by the demand for 18F-fluorodeoxyglucose. The interest in 18F-NaF revived (56–58), and 18F-NaF PET proved to be more accurate and sensitive than 99mTc-diphosphonate SPECT for identifying both malignant and benign lesions of the skeleton in clinical studies (59–62). In contrast to gcamera imaging, PET makes possible the absolute quantification of radioactivity per measured volume. 18FNaF PET allows for a more accurate assessment of the viability of host bone and bone grafts (63), benign metabolic bone disorders (64,65), and the response to bisphosphonate treatment (66). Dual-energy CT is a promising tool for musculoskeletal imaging. It uses two wavelengths of x-ray to scan the object and obtains spatially resolved attenuation value for the two distinct photon energies. The spatial distribution of different chemical compositions can thus be analyzed. The orthopedic applications include imaging of gout crystal, bone marrow edema, tendons, and ligaments, and using monoenergetic techniques to minimize metal prosthesis beam-attenuating ar-

tifacts (67). The sensitivity to detect the rupture of small tendons is still limited. The drawbacks of ionizing radiation–based imaging are highly concerning. Functional imaging using g-ray–generating radionuclides requires the internalization of radioactive isotopes, which may generate health hazards in the long term. Hence, repeated use is restricted. In addition, they have limited half-lives, and the administered dose should be adjusted to balance between the radiation hazard and an adequate signal for imaging. In the following paragraphs, we review the nonionizing imaging modalities for noninvasively studying the bone functions of osteoblastic activity, osteoclastic activity, and mechanical strength. Fluorescence Reflectance Imaging and Fluorescence Molecular Tomography

Fluorescence reflectance imaging (FRI) and bioluminescence imaging (BLI) are important optical imaging techniques and 285

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have previously proved their value in cell and molecular biology studies by providing dynamic imaging of the targeted biomolecules (68,69). FRI and BLI have replaced radionuclide-based imaging in many research settings, and BLI offers even higher sensitivity than FRI because of its higher signal-to-noise ratio (70). FRI, which detects signals from fluorophores concentrated at the site of interest or activated by designated cellular functions, is an important technique for exploring the functional aspects of cells. FRI has become clinical practice in oncology surgery to differentiate healthy tissue from cancerous tissue for its multiplex functional mapping, devoid of ionizing radiation, higher spatial and temporal resolution (71), easier handling, and lower cost. It is intuitive to reconstruct images from ballistic photons, which undergo highly predictable directions of propagation and refraction as well as intensity attenuation, such as radionuclide-based imaging, CT, FRI, and BLI. Because of scattering and absorption, the number of ballistic photons within the visible light and infrared domains decreases substantially after traveling just a few millimeters of biological tissue. Although NIR I/II photons are mildly absorbed by biological tissue, they are highly scattered and become diffuse within
1 mm of their propagation (29) that severely compromises the ability of FRI to map deep objects. Scattering changes the directions of the photons and obscures the sharpness of the images, as with the images that human eyes see in fog. In a diffusely scattering medium, ballistic photons encounter a small number of scattering events and become ‘‘snake’’ photons, which deviate mildly from the direction of the ballistic photons. As scattering events increase and greater deviation occurs, ‘‘snake’’ photons decrease exponentially with depth to become diffuse photons (72). Diffuse optics describes how the light propagates in turbid media and is the basis for diffuse optical spectroscopy and DOT, which is one of Britton Chance’s major contributions (26–31). Diffuse NIR photons can penetrate several centimeters deep in live tissue and are one of the most suitable photons for noninvasive investigation of deep tissue. Xu et al. (73) reconstructed the first absorption and scattered images of human finger bones and chicken bones in turbid media from continuous-wave tomographic measurements in 2001. The volumetric optical images were later recovered using a finite (3D) elementbased reconstruction algorithm (74). Fluorescence molecular tomography (FMT) combines the capability of fluorophore-based molecular imaging and diffuse optics. It has the advantages of low cost and nonionization compared to CT, SPECT, and PET. FMT allows for accurate and quantitative noninvasive imaging of protein concentrations and physiological function and of gene expression. After NIR-emitting fluorophores have been administered, the fluorophores are concentrated at sites of increased vascularity or at selective targeted sites. NIR at the excitation wavelength of the fluorophore is launched into the tissue, and the fluorophores give off NIR at a specific longer wavelength. FMT can be used to map the 3D distribution of fluorophores in two steps. First, photodetector sets or a charge-coupled device 286

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camera is used to make multiple measurements around the tissue surface boundary and to record the pattern of the scattered NIR photons from the fluorescence. Second, tomographic images are reconstructed from an inverse and forward model that predicts the photon distribution arriving at the detectors for a given source location and medium. To enable in vivo applications, Ntziachristos et al. (75,76) proposed the inversion of normalized data to minimize the sensitivity to tissue heterogeneity and to theoretical inaccuracies. The computation is complex in both forward and inverse problems. The need for a fast algorithm is always urgent because the data sets grow in size. Bone formation, resorption, and turnover can be demonstrated by administering bone-seeking fluorescent probes and FMT (77,78). Lambers et al. (79) used a hydroxyapatite-binding fluorescent probe and FMT technology for a longitudinal investigation of bone formation and resorption in mice. The accuracy and sensitivity of FMT for determining bone formation and resorption were compared to micro-CT. The reproducibility was acceptable with a precision error <16%, and the sensitivity was moderate. Optical Imaging of Osteoblastic Activity

Integrating NIR-excitable fluorophores with a targeting construct allows the molecular imaging of deep structures. Zaheer et al. (80) synthesized an NIR fluorescent bisphosphonate derivative using a covalent link between the primary amine group of pamidronate and the N-hydroxysuccinimide ester of NIR fluorophore IRDye78. The derived Pam78 product, with peak absorption at 771 nm and peak emission at 796 nm, rapidly and specifically bound to hydroxyapatite in vitro and in vivo. Optimal images were rapidly acquired (500 ms) 6 hours after a Pam78 injection. Most of the bones in the mice could be visualized using this approach. The skeletal uptake (52%) was greater than that of 99mTc-methyl diphosphonate, and the resolution was also equivalent or higher, except for the deeply located bones. In addition to bisphosphonate-based NIR fluorophores, those conjugated with tetracycline have good bone-seeking properties and can be used as an indicator of bone mineralization and remodeling (81). Optical Imaging of Osteoclastic Activity

The other crucial cellular component for bone homeostasis is the osteoclast, which resorbs bone matrix by acidifying the microenvironment (resorption pits) to demineralize the inorganic component (mainly hydroxyapatite) and then releases the cysteine protease (cathepsin K [CatK]) to break down the organic component (mainly type I collagen) (82,83). Osteoclastic activity is upregulated in illnesses such as Paget’s disease, osteoporosis, malignancy-associated osteolysis, rheumatoid arthritis, and periodontal disease. A loss-of-function mutation of CatK in humans leads to a rare skeletal dysplasia: pycnodysostosis. Mice with deficient CatK have osteopetrosis

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of the long bones and vertebrae, abnormal joint morphology, and abnormalities in hematopoietic compartments (84). Sprague et al. (85) took advantage of the high-level expression of avb3 integrin on the cell membrane of osteoclasts to develop radiolabeled arginyl-glycyl-aspartic acid (Arg-GlyAsp [RGD]), which targets peptide to localize osteoclasts in vivo. In a mouse model of parathyroid hormone–induced osteolysis in the calvarium, PET scans detected increased uptake of the radiolabeled probe. The amount of uptake was linearly correlated with the number of osteoclasts in the bone. Although this technique revealed the abundance of osteoclasts, it was not able to reflect bone resorption activity. Kozloff et al. (86) attempted at revealing the in vivo anatomic distribution of bone resorption activity by developing CatKp, an NIR fluorescence reporter probe. CatKp consists of an MPEG d-poly-lysine amino acid backbone chain functionalized with Cy5.5 fluorophores through the cathepsin K–sensitive link sequence GHPG-GPQGKC, which is relatively insensitive to cathepsins B and L and to metalloproteinase-2 and metalloproteinase-9 (87). In its native form, the fluorophores were optically quenched because of their proximity to one another. Once they had been cleaved, however, the fluorophores were released and were fluorescent. Probe activation was demonstrated in live osteoclasts in vitro and in the murine models of bone development and ovariectomyinduced osteoporosis. In the ovariectomized mice, CatKp activation preceded bone loss detectable by micro-CT. Kowada et al. (88) used the characteristic low-pH environment of the resorption pit, where active bone resorption occurs for osteoclastic imaging. They invented the chemical probes BAp-M and BAp-E (with acid dissociation constants of 4.5 and 6.2, respectively). The probes do not emit fluorescent signals at physiological pH, but they do emit visible light when they have been acidified. The probes contain a bisphosphonate moiety to localize themselves onto the bone. In vivo imaging of osteoclasts was done using twophoton excitation microscopy. Potential clinical applications include using these probes for the early detection of metastatic lesions and as treatment-response monitors for osteoclastic suppression therapy. Effective measures can thus be initiated before significant bone loss. Raman Spectroscopic Imaging

C. V. Raman was the first to describe the wavelength shift of scattered light, for which he won the Nobel Prize for Physics in 1930. The wavelength shift was the result of the inelastic scattering of photons via interaction with the vibrational modes of the molecules (89). Raman spectroscopy belongs to vibrational spectroscopy. The Raman effect is very weak (90). Lasers provide a powerful light source for studying Raman spectra. Every particular substance has its own characteristic Raman spectrum ‘‘fingerprint’’, the intensity of which is linearly proportional to its concentration. This property is useful in nondestructive analytical chemistry. Raman spectroscopy, as well as infrared spectroscopy, is thus an excellent

tool for studying slices of bone matrix ex vivo. Compared to infrared spectroscopy with an attenuated total reflection head (91), Raman spectroscopy has the advantage of being noncontact, little interference with water when using visible light wavelengths, and better spatial resolution (as high as 1 mm, which is at least 10 times greater than infrared spectroscopy) (90). However, the visible-light photons used in Raman spectroscopy could produce significant autofluorescence and hinder detection of the weak Raman signals. Slicing the specimen thin enough to make it transparent is unnecessary because the light scatters rather than being absorbed. For hard tissue, Raman spectroscopy has been used to study the biocompatibility of prostheses and implants, teeth, and archaeological specimens (89). The mineral–matrix ratio, mineral crystallinity, and crosslinking within the organic phase due to genetic change, aging, or microdamage can also be depicted (92,93). One point of spectroscopic data does not represent a whole heterogeneous tissue like bone. Hyperspectral Raman images can be obtained by integrating two-dimensional spatial coordinates with two-dimensional spectral information (wavelength and intensity). The progress in the deep noninvasive characterization of biological tissue derived from DOT presented the opportunity to investigate bone matrix in vivo despite centimeters of intervening soft tissue. This technique is based on spatially offset Raman spectroscopy and transmission Raman (94,95), which extend the depth of conventional profiling from hundreds of micrometers to several millimeters or even centimeters in some cases. This technological advance takes advantage of suppressing intense interfering Raman signals and fluorescence signals from the near-surface area (96). An excellent review of noninvasive deep Raman spectroscopy in medical applications is available (97). A crucial function of bone is mechanical support. Bone strength depends on bone geometry and bone quality, and bone quality is determined by the organization of its mineral and organic parts. The noninvasive imaging modalities to study the ‘‘supporting function’’ of bone are summarized in Table 3. The current standard clinical tool for determining bone quality is dual-energy x-ray absorptiometry (DXA). DXA as a bone mineral densitometer measures primarily the amount of mineral component of the bone and ignores the organic component. The accuracy of DXA alone to predict osteoporotic fracture is limited, which stresses the mechanical function of the organic component. There are detectable differences in collagen crosslinks between healthy and osteoporotic bone (98,99). The first noninvasive assessment of bone matrix using a time-gated Raman spectrum was done in OIM/OIM mice (a murine disease model for osteogenesis imperfecta) in 2005 (100). In 2006, the first transcutaneous Raman spectrum of the human distal phalanx of the thumb in vivo was obtained at skin-safe laser illumination levels (96). The Raman spectrum of the deep tissue was collected by concentric rings of optical fibers away from laser illumination. Schulmerich et al. (101) used transcutaneous Raman spectroscopy on the bones of rats and chickens; the 287

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288 TABLE 3. Current Noninvasive ‘‘Bone Quality’’ Imaging Modalities

Modality

Dual-energy X-ray Absorptiometry

Quantitative Computed Tomography

Quantitative Ultrasound

High-resolution Magnetic Resonance Imaging

Raman Spectroscopic Imaging

Bone mineral density per unit projection body surface area

1. Bone mineral density per voxel 2. Trabecular geometry

1. Speed of sound 2. Broadband ultrasound attenuation 3. Stiffness index 4. Bone transmission time 5. Quantitative ultrasound index (These variables are influenced by both bone density and architecture)

1. Trabecular parameter (Requires higher magnetic field, like 3 T) 2. Water content 3. Organic bone matrix density 4. Bone mineral content (measured using phosphorus spectroscopy) 5. Mineral-to-matrix ratio

1. Mineral-to-matrix ratio 2. Carbonate-to-phosphate ratio 3. Collagen quality (crosslinking) 4. Crystallinity

Skeleton assessed

Central (hip and spine)

Central and peripheral

Central and peripheral

Peripheral

Image acquisition time Ionizing radiation exposure

Minutes Low (0.08–4.6 mSv for pencil beam; 6.7–31 mSv for fan beam)

Minutes to hours No

Minutes No

Portability Application

Poor Clinical standard

Minutes 1. High (50–100 mSv) for central skeletal evaluation 2. Low (<2 mSv) for peripheral skeletal evaluation Poor Clinical

Peripheral (calcaneus, radius, phalanges, and tibia) Seconds to minutes No

Good Clinical

Poor Research

Moderate Research

Data are integrated from the previous studies (94,226–230).

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Variable derived from bone tissue

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error in the measured carbonate-to-phosphate ratio was <8% with a 120-second integration time for chicken tibia 4 mm below the skin surface. Maher et al (102) developed a new spectral extraction algorithm called simultaneous overconstrained library-based decomposition, which enhanced the mean spectral correlation coefficient among exposed bones from different mice to 0.988 (from 0.935 by widely used band target entropy minimization algorithm) and reached diagnostic sensitivity. Fourier transform infrared (FTIR) spectroscopy is another vibrational spectroscopy highly complementary to Raman spectroscopy in its sensitivity to different chemical structure species. Using the Synchrotron Radiation Lightsource augments the detection sensitivity and spatial resolution of FTIR imaging. FTIR was recently used to map the fossilized bones in a series of dinosaur embryos (103). FTIR imaging has also been widely used to investigate molecular changes in orthopedic diseases, such as osteoarthritis, osteoporosis, osteopetrosis, and osteogenesis imperfecta (104). However, because water molecules have significant absorption spanning the IR wavelengths, practical clinical use of FTIR imaging still requires evaluation and development. Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy for Functional Imaging of Bone

Magnetic resonance imaging, which is based on nuclear magnetic resonance (NMR), is one of the most widely used medical imaging modalities because of its excellent soft tissue resolution and versatile imaging capability. NMR is a physical phenomenon in which the nucleus of an atom with an odd number of protons or neutrons (or both) absorbs and reemits electromagnetic radiation at the radiofrequency domain. MRI generates images of anatomic structures and pathologic lesions by delineating differences in the density, spin–lattice (T1), and spin–spin (T2) relaxation time of the hydrogen nuclei. The relaxation times are sensitive to fluid flow, temperature, water and lipid content, molecular motion, the chemical environment, and nearby magnetic nuclei. Therefore, more detailed anatomic and functional information of the human body can be obtained from them than from x-ray–based modalities. The study of calcified or mineralized tissue in vivo, which normally used CT, has gradually progressed to solid-state MRI (ssMRI) that is capable of examining the topologic, geometric, and chemical orders of hard tissue (105). ssMRI has become a promising tool for assessing the structural, functional, and physiological aspects of bone, for evaluating bone developmental and repair (106,107). Blood-oxygen-level–dependent (BOLD) MRI introduced in 1990 is a valuable tool for studying fiber types and the vascular response of muscles to diseases, such as muscular dystrophy, ischemia or chronic venous insufficiency, and Parkinson’s disease (108). The principle of BOLD lies in alterations in the ratio of diamagnetic oxyhemoglobin to paramagnetic deoxyhemoglobin in the microvascu-

lature. Inflammation in biological tissue, as a functional event, leads to an increased water content of the tissue, which can be detected by noncontrasted or static and dynamic contrastenhanced MRI (109). Molecular imaging using MRI is also feasible. The probe for MRI of molecular events is typically an activatable probe (110,111) or a probe synthesized by conjugating MRI contrast agent with a proper targeting probe like bisphosphonate derivatives, a portion of an antibody, a peptide, a receptor ligand, or another small molecule. The options for the MRI contrast probe include Gd3+ chelates (112–114), Gd NPs (115), magnetic NPs, and metalloproteins (116). Magnetic resonance spectroscopy (MRS), also known as NMR spectroscopy, is used to provide functional information by profiling the distribution and quantity of the metabolites in the musculoskeletal system (117), cardiovascular system (118), neuroscience (119), oncology (120,121), stem cell research (122), and so on. In addition to hydrogen nuclei, spectroscopic imaging of 31P, 23Na, 19F, and 13C nuclei reveal more functional information. Britton Chance was a pioneer in the MRS imaging (123) of energy metabolism of the muscle, brain, heart, and tumors (117,120,124–128). Gade et al. (111) imaged osteoblastic activity in vivo by measuring alkaline phosphatase enzyme activity using 19fluorine MRS imaging. They designed a small-molecule substrate for alkaline phosphatase (6,8-difluoro-4-methylumbelliferyl phosphate). The designed substrate has two fluorine atoms adjacent to a phosphate group, which allows the hydrolytic product to be distinguished from the native substrate. MRI also has an emerging application in quantifying bone quality (Table 3). The trabecular parameters can be derived in high-resolution MRI. The bone mineral content can be measured using phosphorus spectroscopy and the organic bone matrix density using water- and fat-suppressed proton projection MRI(129). The ratio of bone mineral content to organic bone matrix density reflects the extent of the mineralization of the bone, which is critical for diseases, such as osteomalacia and rickets. The advantages of MRI include high anatomic resolution with unlimited depth of access, absolute quantification via spectroscopy, and multiple choices of imaging sequences for many sorts of information. The major drawback of conventional MRI is the limited temporal resolution and low sensitivity to a molecule of interest. Ultrasound for Functional Imaging

Ultrasonography is characterized by high temporal (real-time) and spatial resolution at low cost and is an important tool in orthopedic clinics. The disadvantage is frequencydependent limited depth access and blockage by gas and hard materials, such as lithiasis (stone), calcification, or bone. Hence, its application in the musculoskeletal system is mainly for imaging muscle, tendon, nerve, joint, and bone surface. The mechanical properties of bone change ultrasound transmission parameters, and quantitative ultrasound is capable of measuring bone quality (Table 3). Inflammation289

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induced fluid accumulation is readily visible in ultrasonography (109). Doppler ultrasound is used to detect blood flow and vascularity changes in conditions like tumor, overuse, or rheumatologic diseases (109,130,131). A recent development in ultrasound is sonoelastography, which detects tissue elasticity and is helpful for distinguishing pathologic lesions with echogenicity similar to that of the surrounding tissue, for example, edema, hemorrhage, progressive tendinosis or mucoid degeneration, and partial tears in the tendon (132). Microbubbles, gas-filled particles with an average diameter of several micrometers (133), were developed as a contrast agent for ultrasonography. Microbubbles in the blood vessels undergo volume oscillations at clinical diagnostic ultrasound imaging frequencies (1–15 MHz) to generate a backscatter ultrasound signal that can be detected. It offers a high signal-tonoise ratio to facilitate detection of low-volume blood flow in small vessels. Microbubble contrast-enhanced ultrasonography was first reported to assess myocardial perfusion (134). Its current clinical applications are limited to echocardiography and hepatocellular cancer imaging. Molecular imaging using ultrasound is enabled by microbubbles functionalized with targeting ligands. These particles are injected intravenously to circulate blood vessels and then bind to target receptors on vascular endothelium. The particles reflect ultrasound to allow visualization of the biomarkers in the anatomic site. A successful small-scale clinical trial using microbubbles to target tumor vasculature (135) encouraged further development of microbubble-enabled molecular imaging. The current major limitation of functional ultrasonography using targeting microbubbles is the confinement of molecular targets to vascular endothelial surfaces, which makes it relatively slow. NANOMEDICINE-ENABLED IMAGING IN ORTHOPEDICS Imaging modalities and molecular probe designs for mineral tissue have significantly improved within the past decade. In addition to their distinctive functional properties, NPs share a comparable size range close to biomolecules. Nanomaterials for Magnetic Resonance Imaging Probes

Magnetic NPs consisting of iron, nickel, cobalt, and their chemical compounds have super paramagnetic or ferromagnetic properties; thus, they perturb local magnetic fields. They shorten the T2 relaxation time and have a negative contrast effect (low signal) in T2-weighted MR sequences (136). SPIONs are most commonly used as MRI contrast agents. Biocompatible SPION well dispersed in the aqueous phase are essential for their biomedical applications. Sixnanometer SPION with an amine group surface modification, synthesized using bioorganic acid, was selectively ingested by Kupffer cells in the liver to detect hepatoma lesions in clinical MRI (137,138). The advantages of SPION 290

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as an MRI contrast agent include adjustability of T1 and T2 relaxivity, biodistribution, and targeting function by changing their particle size and surface chemistry (139,140). Larger particles are used as bowel contrast agents and for liver and spleen imaging. Smaller particles are suitable for angiography and imaging lymph nodes, perfusion, and bone marrow. Smaller monocrystalline particles are also good candidates for receptor-directed MRI of selective cell types and target molecules. SPIONs have been engineered as bone-seeking probes. The system bisphosphonates to implement bone-seeking property, such as the 99mTc-diphosphonates, but without the risk and extra cost associated with ionizing radionuclides. Panahifar et al (15) reported a relatively simple, quick, and cost-effective in vitro method for conjugating SPION to alendronate while preserving the functional phosphate group for hydroxyapatite binding, where the functional phosphate group is known to complex strongly with iron (141). Lalatonne et al. (142) conjugated gFe2O3 SPION with 1,5-dihydroxy-1,5,5-tris-phosphonopentyl-phosphonic acid. The SPIONs were not cytotoxic in vitro. They adsorbed to hydroxyapatite and were used as MRI T2* contrast agents in vitro. In vivo MRI imaging for bisphosphonate-conjugated SPION is pending. Molecular MRI is also a powerful tool for investigating stem cell migration and immune cell trafficking (143). However, because the NPs were excreted and, therefore, could be transferred from the original target cells to nontarget populations, probe design and interpretation of the observation are critical. Another concern is the effect of SPIONs on the ‘‘stemness’’ of the labeled cells. In a recent study (144), this concern was addressed by a series of analysis of SPIONlabeled versus nonlabeled bone marrow stem cells using colony forming efficiency, CD146 expression, gene expression profiling, and bone and myelosupportive stroma formation in vivo in immunocompromised recipients. The results indicate that SPION-labeling did not change their ‘‘stemness’’. Jing et al. (145) used SPION probes with 1.5T clinical MRI for up to 12 weeks to track bone marrow–derived mesenchymal stem cells (MSCs) injected into the intra-articular space of rabbit knee joints. The T2-weighted image showed marked negative contrast enhancement of the implanted MSCs in the intra-articular space that lasted for at least 12 weeks; signal intensity peaked in the fourth week. Microscopic examination revealed that the SPION was retained in the cytoplasm of the labeled cells and that significant proteoglycan and type II collagen had been deposited, which showed their chondrogenic functions. Another study (146) discovered that the U.S. FDA-approved ferumoxide (a type of SPION) and protamine could be combined to improve the uptake efficiency and retention of SPION by MSCs for MRI tracking. Gold NPs in Computed Tomography and Photoacoustic Imaging

Gold NPs are one of the earliest synthesized nanomaterials in the world dated centuries ago. The biocompatibility and

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chemically inert state of gold NPs are excellent for diagnostic and therapeutic applications. Furthermore, it was first reported in 2006 (147) that 1.9-nm gold NPs showed great potential as an x-ray or CT contrast for their higher attenuation than iodinated contrast, the current standard CT imaging contrast. The gold NPs were excreted by the kidneys, and their in vivo biosafety was shown in the 30 days of follow up. Gold NPs interfere less with bone and soft tissue than does iodinated contrast in 80–100 keV x-rays, which allows better contrast with a lower x-ray dose. Gold NPs also have a longer circulation period, which permits a longer observation time (>24 hours) (148), which is useful for CT-guided interventional procedures. Processing gold NPs as CT contrast or molecular probes has been reported in many previous studies (149–153). Thiol, disulfide, or other sulfur derivative (dithioester and trithiocarbonate) terminated polymers or organic compounds have been used to form stable gold–thiol bonds on gold NP surfaces for biological stealth, targeting, or other biological functions. Bonetargeting gold NP probes are made by conjugation with bisphosphonates or L-glutamate (16,18). Such designs are still confined to in vitro study, however, and have to be further explored to confirm their clinical practicality. Besides the high electron density and thus x-ray attenuation applicable for x-ray imaging, gold NPs have distinct localized surface plasmon resonance (LSPR) peak wavelengths proportional to their geometric aspect ratio. Because the resonance wavelengths of gold nanorods fall within the NIR biological window spectra domain, gold nanorods have attracted significant attention for potential biomedical imaging and therapeutic applications due to the relatively deep transmissibility of NIR photons into biological tissue (154). Because the optical properties of gold nanorods are tunable based on particle size, shape, and dielectric environment, multiplex molecular imaging is made possible by using different species of gold nanorods for multiple target biomolecules (155–159). In addition to their optical absorption, gold nanorods provide scattering contrast for dark field microscopy, and also emit a strong two-photon luminescence because of plasmon-enhanced two-photon absorption (160). The advantages of gold nanorods as a photoluminescent probe include resistance to photobleaching and greater linear and nonlinear absorption cross sections than organic fluorophores (160). Optical coherence tomography typically generates images based on morphology-dependent scattering, differential absorption contrast (spectroscopic mode), or differences in absorption-scattering profiles. Gold nanorods as a modulator in optical absorption or scattering within an NIR domain can be the contrast of choice for optical coherence tomography (161,162). Light absorption can be alternatively followed predominantly by heat generation because of nonradiative electron relaxation dynamics (163), and the released heat and subsequent temperature rise can thermally injure adjacent cells or cause a strong photoacoustic (optoacoustic) effect. Photoacoustic imaging (PAI) is a hybrid, nonionizing modality, which is of particular interest because of the much higher

spatial resolution and good soft tissue contrast. PAI relies on the intrinsic differences in optical absorption within the tissue being imaged. NIR-absorbing gold nanorods have recently been used in vivo as contrast agents for PAI to increase the signal intensity for molecular imaging of oncogene expression in tumor lesions (155). Simultaneous ultrasonography and molecular PAI of tumor angiogenesis blood vessels and oncogene expression in tumor cells was used in a tumor-bearing mouse model. The same molecular probes were applied to derive a significant regression of tumor growth in vivo by laser-induced hyperthermia. Other applications include quantitative flow analysis in biological tissue (164) and distribution kinetics of drug delivery systems (165). Other forms of gold NPs (e.g., gold nanocages) have also been synthesized and used as a multimodality imaging or theranostics platform because their LSPR peaks can readily and precisely be tuned to any NIR wavelength (166). Multimodality contrast can be made by radiolabeling with 64Cu for PET (167) or with 198Au for Cerenkov luminescence imaging (168). Other Optically Active Nanomaterials for Optical Imaging

Colloidal crystalline semiconductor NPs (quantum dots) with their three dimensions <10 nm present unique photophysical properties (169). The bright photoluminescence effect of quantum dots is generated by the combination of high quantum yields and large molar extinction coefficients. Quantum dots have wide absorption spectra but narrow emission spectra. By tuning the temperature, duration of crystal growth, ligand molecules used during synthesis, different sizes of quantum dots, and thus different photoluminescence colors, can be obtained. Other excellent optical properties of quantum dots compared to fluorescent dyes include extended excited-state lifetimes (>10 ns), superior resistance to photobleaching and chemical degradation, and orders of magnitude greater two-photon absorption cross-sections (103–104 GM) (170). The resistance to photobleaching is especially useful for 3D optical sectioning, for which a major issue is fluorophore bleaching during the acquisition of successive z-sections, which significantly compromises correct 3D anatomy reconstruction. The brightness and resistance to chemical degradation makes quantum dots ideal candidates for animal molecular imaging. Larson et al. (170) showed that higher contrast and imaging depth could be obtained at a lower excitation power than with organic dyes when mice intravenously injected with quantum dots were subjected to two-photon excitation confocal microscopy of their blood vessels. Live animal imaging of targeting quantum dots is achieved using antibody-conjugated quantum dots and NIR-emitting quantum dots (171). Cytotoxicity and the interference with cellular processes are the main concerns for quantum dots in live-cell or animal experiment. Advanced surface modification and structural engineering of the quantum dots seemed to affect its toxicity and metabolic clearance that is essential for further clinical development (171). 291

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Mesoporous silica NPs (MSNPs) were developed as a multifunctional platform to deliver diagnostic and therapeutic agents because they are adequately biocompatible, stable, and versatile enough to be nanocarriers (172). MSNPs have been extensively developed for diagnostics and theranostics in the field of bone and tendon tissue engineering, infectious diseases, diabetes, inflammation, and cancer in the past decade (173). They have been used to encapsulate therapeutic agents, SPIONs, gadolinium, and fluorescent dyes and to assemble targeting groups on the surface. Huang et al. (174) discovered that FITC-MSNPs are excellent for cell labeling and do not affect cell viability, growth, or differentiation. The particles are resistant to lysosomal degradation and are retained longer in cells than does free fluorochrome. The results suggest the potential niches of MSNPs in mesenchymal stem cell tracking (175). Modifying the porous structure with gadolinium, however, enables the MRI tracking function of the particles (176). Integrating the paramagnetic and fluorescent labeling, van Schooneveld et al. (177) developed multimodal silica NPs capable of imaging contrast in MRI, CT, and fluorescence imaging. Although silica-based NPs have been used in bone bioengineering and to stimulate bone formation, its use in bone imaging is still in its infancy (178,179). Organic Materials for Bone Imaging

Organic NPs have also been used to improve the targeting function and biodistribution of molecular imaging probes. Park et al. (180) reported the advantage of extended circulation time and high bone uptake of 100-nm 125I-labeled poloxamer 407-poly(lactic-co-glycolic) acid (PLGA) NPs in whole-body imaging. In addition to high labeling efficiency (90%) and stability in human serum as well as decreased liver and spleen uptake, these NPs are fully biodegradable and are thus a promising tool for bone-targeting molecular imaging contrast (181). Biological molecules are in the size range of one nanometer to tens of nanometers. Certain structural features of biological macromolecules present distinct linear and nonlinear optical properties and thus enable noninvasive imaging in the human body. For example, the oxygenation status of hemoglobin induces a shift in its optical absorption in the NIR region, which makes possible the PAI of tissue oxygenation and the mapping of arterial versus venous vascular networks in 3D (181–183). Type I collagen molecules exhibit a strong second order nonlinear susceptibility, which translates into a strong second harmonic generation (SHG) signal (184,185). Because type I collagen is the major organic component of osseous tissue, noninvasive imaging of collagen and the osseous mineral component would provide valuable information to disease diagnosis. Figure 2 presents the first attempt at integrating the SHG and the third HG (THG) imaging (186,187) of a human fibrous dysplasia lesion. In the optical sections of the lesion, the SHG signal from collagen fibrils and the THG signal from cellular components (188), as well as the osteoid tissue, are clearly visible. In the two serial-sections, the lamellar structure and the lacuna of the 292

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osteoid mineral deposition are identifiable. Harmonic generation imaging has the significant advantages of high spatial resolution, real-time 3D tomographic imaging, high tissue penetrability because the second biological optical window is used as the light source, and causing the least photon damage to tissue because energy deposition is in a virtual state (189). Further translational development is encouraged. Upconversion NPs

Upconversion NPs exhibit permanent, strong, anti-Stokes emission of discrete shorter wavelengths of light when excited by two or more photons at longer wavelengths (usually at NIR or IR domain) (190). Upconversion emission involves excited-state excitation, energy transfer upconversion, and a photon avalanche (190). Compared to conventional antiStokes processes, such as SHG and multiphoton absorption, upconversion emission is facilitated by physically existing states, which offer efficient frequency conversion. Thus, upconversion processes can be induced by a low-power (1– 103 W/cm2) continuous wave laser, in contrast to a costly high-intensity (106–109 W/cm2) pulsed laser source, to generate a simultaneous two-photon process (191). Lanthanide-doped upconversion nanoparticle (UCNP) size, crystalline phase purity, morphology, and monodispersity determine upconversion efficiency and emission wavelength, which enables multicolor imaging (192,193). Most UCNPs consist of an inorganic particle that is doped with lanthanide as a sensitizer and activator (194). The advantages of using UCNPs (195–197) include increased depth penetration into tissue on NIR excitation, significant decrease in the autofluorescence from surrounding tissue, nonphotobleaching and nonphotoblinking (198), high spatial resolution during bioimaging, decreased photodamage to biological specimens (e.g., RNA, DNA) because of lower energy NIR excitation, and low cytotoxicity in a broad range of cell lines (199,200). Such special upconversion luminescence makes UCNPs promising in fluorescence bioimaging, biological sensing and detection, developing point-of-care devices, and drug delivery. UCNPs are also an attractive component of composite nanostructures for multimodality imaging (193). Zijlmans et al. (201) reported the first UCNP luminescence system in 1999 to detect prostatespecific antigen expression on cell surfaces and in tissue sections. In vivo imaging of Caenorhabditis elegans using UCNPs was pioneered by Lim et al. (202). In 2012, Chen et al. (203) proposed that such UCNPs can be visualized as deeply as in rat femoral bones wrapped in a 3.2-cm–thick tissue of pork. DEVELOPING FUNCTIONAL IMAGING TECHNOLOGIES The Modular Design in Targeting

The major obstacles for NP-enabled targeted MRI include effective bioconjugation, in vivo pharmacokinetics, and the

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Figure 2. Integrated second harmonic generation (SHG) and third harmonic generation (THG) imaging of a human fibrous dysplasia lesion. Serial horizontal optical sections (a–d) of a fibrous dysplasia lesion show an intense SHG signal (green) from collagen fibrils and a THG signal (red) from the cellular component and the osteoid tissue. The lamellar structure of the mineralizing osteoid is indicated by an arrow. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.

sensitivity of the detection. Orientation control of targeting moieties on the particle surface is a challenge using traditional chemical conjugation strategy because multiple sites on a given ligand might present the same target function group. Moreover, cross-linking of molecular probes also compromises their effective targeting and biodistribution. Multiplex molecular imaging makes it possible to visualize protein or gene expression profiles in vivo. However, conjugating an individual targeting moiety to different reporter NPs is tedious and complicated. To solve these issues, Shieh et al. (204) presented the concept of modular design for fabricating NPs by engineering a self-assembling chemical process to link the functional reporter part of the NP to the targeting part of the engineered antibody. Multimodal Imaging Contrast Agents

Multimodality imaging like PET/CT, SPECT/CT, and PET/MRI has not only gained clinical success in advanced disease diagnosis but also become a rapidly evolving field in experimental research and translational development. It is a high-interdisciplinary field combining oncology, neurology, psychiatry, cardiology, molecular biology, cell biology, pharmacology, nanotechnology, physics, and engineering. Every imaging modality has its unique advantages and intrinsic

weaknesses or limitations. Multimodality imaging obtains and integrates complementary information (structural and functional imaging) from a subject at the same examination session to define specific functional events in a synchronized way with high anatomic and temporal relevance. Combining different imaging modalities offers synergistic benefits in imaging itself. Combining SPECTor PETwith CT allows image enhancement using the anatomically registered information of attenuation correction obtained from CT. The spatial resolution of PET in PET/MRI is promoted because of the decreased positron range under strong magnetic field (205). Ultrasonographic and PAI systems can be integrated because the acoustic signal from both systems can be acquired by a common detector. Xu et al. (206) used such a system to image human peripheral joints. Inflammation in the joint was detectable by the photoacoustic system because of synovial neovascularization (207). The advantages of noninvasive multimodality imaging include increased sensitivity and accuracy in disease screening, staging, and treatment-response evaluation as well as drug–target interaction, cell-tracking, and pathophysiological-mechanism investigation. Different aspects of information concerning the patient, characteristics of the disease, and the microenvironmental condition can be noninvasively collected and longitudinally investigated, which is valuable for personalized medicine. 293

Probes Gold nanoparticles

Imaging Modality Fluorescence microscopy or FRI Dark-field microspectroscopy Optical coherence tomography CT MRI contrast enhancer

PAI Surface-enhanced Raman spectroscopy

MRI

Plasmon-based photoluminescence Strong and characteristic light scattering Strong light scattering Photon absorption 1. Carry multiple Gd chelates 2. Increase magnetism of Co@Pt-Au NP Strong light absorption Huge increase of the Raman signal, by a factor of about 1014 to 1015 to allow single molecular detection

Disturbance of magnetic field

Advantages/Disadvantages Advantages 1. Good photostability 2. Tunability of wavelength absorption/scattering coefficient by its size, shape, aggregation, and medium to increase tissue penetration 3. Good biocompatibility (can protect and increase biocompatibility of other NPs as their coating) 4. Longer circulation time than iodine CT contrast 5. Higher x-ray photon absorption than iodine contrast Disadvantages 1. Relatively low quantum yield 2. Moderately toxic for cationic gold NP Advantages

Clinical and Research Implications

References

(154,157,231) 1. Most clinically relevant imaging applications act as CT-contrast and MRI-contrast enhancer, which provides a better contrast effect than does a traditional contrast agent 2. Strong photoacoustic effect makes gold nanorods a promising theranostic platform 3. May find use in dermatology and ophthalmology as a good contrast for optical coherence tomography 4. The optical property allows only superficial imaging 5. Other characteristics are versatile for research and biosensor use

1. MRI as the only nonionizing (139,140,232) whole body imaging 1. Good biocompatibility modality in clinical use 2. Deep access allows 2. Clinical applications include whole body imaging cell tracking, passive tar3. Tunability of biodistribution geting, and active targeting and excretion by its size and 3. Passive targeting enables surface modification. liver/spleen imaging, 4. Approved for clinical use macrophage imaging, under specific indication lymph node imaging, and 5. Magnetic relaxation cancer imaging switching (MRS) increases 4. Active targeting enables sensitivity cancer imaging, apoptosis Disadvantages imaging, and cardiovascular 1. Relatively low sensitivity imaging in MR-based imaging 5. MRS enables detection of 2. Incapable of multiplexed oligonucleotides, proteins/ imaging enzymes, and enantiomers

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Superparamagnetic iron oxide NP

Mechanism

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TABLE 4. Comparison of Nanoparticle (NP) Imaging Probes for Functional Imaging in Clinical and Research Use

Optical imaging

Quantum confinement effect

Advantages 1. Wide range of size-tunable light emission (from ultraviolet to NIR II) 2. High molar extinction coefficient and quantum yield (high brightness) 3. Photostability 4. Large Stokes shift (good signal-to-noise ratio) 5. Simultaneous excitation of multiple fluorescence colors (based on broad absorption spectrum and narrow emission spectrum) Disadvantages

1. Excited-state excitation 2. Energy transfer upconversion 3. photon avalanche

(169,171) 1. Multicolor imaging allows simultaneous tracking of multiple molecular targets 2. The better optical properties and adjustability allows deeper access than gold NP 3. Traditional semiconductor QD cores are highly toxic but biocompatible QDs have been developed

1. Potential cytotoxicity due to exposure of some inorganic core (eg, cadmium) 2. Blinking (191,193,195) 1. Most favorable optical Advantages properties and adjustability 1. Good biocompatibility allow the deepest tissue 2. Large anti-Stokes shift access. (UCNP with NIR II in some absorption and NIR I 3. Photostability emission is feasible) 4. Low background noise 2. Theranostic platform could (tissue autofluorescence) be built on UCNP for its 5. Low input light power deep imaging capability and needed upconverting emission of 6. Tunable emission light ultraviolet light wavelength (ultraviolet 3. Low input light power cause to near-infrared) less tissue photodamage Disadvantages 4. Still insufficient for whole Low quantum yield, especially body imaging small-size UCNP for intracellular application

CT, computed tomography; FRI, fluorescence reflectance imaging; MRI, magnetic resonance imaging; NIR, near-infrared; PAI, photoacoustic imaging. Common charateristics for NPs listed in the table: (1) surface modification for multiple biological and chemical functions, (2) integrated properties for multimodality imaging, (3) good photostability, (4) adaptive physicochemical and biological properties for various imaging modalities.

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Lanthanide-doped upconversion nanoparticle (UCNP)

Fluorescence reflectance imaging

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The full potential of multimodality imaging is augmented by multimodality contrast agents and targeting probes. Distinct properties of multifunctional nanomaterials make them feasible for targeted multimodality imaging contrast agents and as theranostic platforms (208–210). Advances in synthesizing alloy NPs have enabled multimodality imaging contrast agents. Shieh et al. (211) synthesized water-soluble FePt NPs with excellent biocompatibility. The tunable size of the NPs led to different biodistributions. The anti-Her2/ neu antibody-conjugated FePt NPs showed a molecularexpression–dependent CT/MRI dual-imaging contrast effect in a cell-line study and in a tumor-bearing animal model. Stemming from the potential for radiofrequency-induced hyperthermia, this NP is also promising for future theranostic platforms (212,213). Agarwal et al. (214) invented a contrast agent combining gold nanorods and tumor necrosis factor-a antibody (TNF-a Ab) radiolabeled with 125I that yielded a good correspondence between photoacoustic and nuclear imaging: PAI provided sub-millimeter spatial resolution, and nuclear imaging sensitively detected TNF-a Ab delivery. Shao et al. (215) devised an inflammation-targeted nuclear and optical dualmodality contrast agent prepared by 125I-radiolabeled gold nanorods conjugated with anti–intercellular adhesion molecule 1 (ICAM-1) antibody, and demonstrated increased ICAM-1 expression in the affected ankles of rats with adjuvant-induced arthritis. Multimodality imaging contrast agents provide a spatial reference for different imaging modalities. A clinical problem in orthopedics is distinguishing, when using plain radiography, CT, and MRI, compositionally similar calcium phosphate cement (a commonly used bone substitute) from native bone. Ventura et al. (216) solved the problem with silica beads that carry contrast-enhancing NPs—colloidal gold and SPIONs—incorporated within a calcium phosphate powder to significantly increase contrast enhancement.

CONCLUSION Functional imaging is becoming increasingly important in clinical medicine because it detects functional and molecular changes of a disease, which in most circumstances precede the structural changes that traditional imaging techniques detect. Noninvasive functional imaging of bone is challenging because of the high mineralization and deep location of human bone in vivo. Abundant ballistic g-ray photons can be easily obtained from deep live tissue after an intravenous injection of radionuclides because of the very low absorption and scattering of these high-energy photons. Hence, nuclear medicine was established as the gold standard in orthopedic functional imaging after its FDA approval in 1972. The introduction of 18 F-NaF PET further promotes the sensitivity, accuracy, and timesaving qualities compared to 99mTc scintigraphy and SPECT. SPECT has made a significant progress in imaging 296

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resolution by improving the collimator design. The clinical value of functional imaging using SPECT and PET increases when a structural-imaging modality, such as CT or MRI, is added, which results in multimodality imaging. With the development of nanotechnology, novel and promising multimodality imaging contrast agents have been developed. Scattered or diffuse photons have long been considered noise and interference, which render the accurate measurement of deeply buried objects impossible. Thanks to the brilliant mind of Britton Chance, these nuisance diffuse photons have been transformed into valuable reporter signals. Fluorescence molecular tomography can now quantitatively image NIR-emitting probes at picomolar concentrations several centimeters deep in live animals. The development of diffuse optics also enables Raman spectroscopy, a prospective tool for bone-quality analysis and for subsurface probing. Another significant contribution to biomedical imaging from Chance is MRS, for which he won the gold medal from the Society of Magnetic Resonance in Medicine in 1988. MRS enabled the noninvasive investigation of muscle dynamics, brain injury and metabolism, and tumor biology. NPs with distinctive properties of magnetism, optics, acoustics, and surface chemistry have been extensively explored as imaging probes, vectors for drug delivery, and even theranostic platforms in multiple clinical specialties; however, their use in orthopedics is still rudimentary. The characteristics, clinical, and research implications of different NP-based probes are provided in Table 4. Molecular imaging with NPs is facilitated by their favorable surface chemistry after they have been conjugated with a targeting unit. Multiplex imaging is based on the tunable physical properties of NPs, depending on their nanoscale size and shape, which can be manipulated during the manufacturing process. The tedious process of conjugating the targeting unit to different reporter NPs can be simplified by using a modular design. The upconversion NP as a promising deep biomedical probe because of its biocompatibility, photostability, low background autofluorescence noise, and low-power laser input is noteworthy. However, the quantum yield is still not high enough for smallsize upconversion NP. Significant progress has been achieved in microscopic and small-animal imaging for the last decade, but only few have been translated into regular clinical practice. Current technologies for whole-body optical imaging of deep human tissue are far from satisfactory. A major obstacle is the huge size of the human body relative to that of small animals. The challenges include: (1) brighter biocompatible optical probes with lower biological autofluorescence and lower input energy, (2) more sensitive detectors at specified wavelength ranges, (3) more accurate yet computable modeling of biological tissue, (4) greater computational power, (5) smaller overall instrument size for portability, and (6) affordability. With the current rapid pace of multidisciplinary development and integration, challenges are turning into opportunities, and Britton Chance’s dream of using optical imaging in multiple fields of clinical medicine will undoubtedly be realized in the near future.

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