Jacqueline H. Cole

Noninvasive Raman tomographic imaging of canine bone tissue.

Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images.

Transcutaneous Raman spectroscopy of murine bone in vivo.

Raman spectroscopy can provide valuable information about bone tissue composition in studies of bone development, biomechanics, and health. In order to study the Raman spectra of bone in vivo, instrumentation that enhances the recovery of subsurface spectra must be developed and validated. Five fiber-optic probe configurations were considered for transcutaneous bone Raman spectroscopy of small animals. Measurements were obtained from the tibia of sacrificed mice, and the bone Raman signal was recovered for each probe configuration. The configuration with the optimal combination of bone signal intensity, signal variance, and power distribution was then evaluated under in vivo conditions. Multiple in vivo transcutaneous measurements were obtained from the left tibia of 32 anesthetized mice. After collecting the transcutaneous Raman signal, exposed bone measurements were collected and used as a validation reference. Multivariate analysis was used to recover bone spectra from transcutaneous measurements. To assess the validity of the transcutaneous bone measurements cross-correlations were calculated between standardized spectra from the recovered bone signal and the exposed bone measurements. Additionally, the carbonate-to-phosphate height ratios of the recovered bone signals were compared to the reference exposed bone measurements. The mean cross-correlation coefficient between the recovered and exposed measurements was 0.96, and the carbonate-to-phosphate ratios did not differ significantly between the two sets of spectra (p > 0.05). During these first systematic in vivo Raman measurements, we discovered that probe alignment and animal coat color influenced the results and thus should be considered in future probe and study designs. Nevertheless, our noninvasive Raman spectroscopic probe accurately assessed bone tissue composition through the skin in live mice.

Whole Bone Mechanics and Bone Quality

BackgroundThe skeleton plays a critical structural role in bearing functional loads, and failure to do so results in fracture. As we evaluate new therapeutics and consider treatments to prevent skeletal fractures, understanding the basic mechanics underlying whole bone testing and the key principles and characteristics contributing to the structural strength of a bone is critical.Questions/purposesWe therefore asked: (1) How are whole bone mechanical tests performed and what are the key outcomes measured? (2) How do the intrinsic characteristics of bone tissue contribute to the mechanical properties of a whole bone? (3) What are the effects of extrinsic characteristics on whole bone mechanical behavior? (4) Do environmental factors affect whole bone mechanical properties?MethodsWe conducted a PubMed search using specific search terms and limiting our included articles to those related to in vitro testing of whole bones. Basic solid mechanics concepts are summarized in the context of whole bone testing and the determinants of whole bone behavior.ResultsWhole bone mechanical tests measure structural stiffness and strength from load-deformation data. Whole bone stiffness and strength are a function of total bone mass and the tissue geometric distribution and material properties. Age, sex, genetics, diet, and activity contribute to bone structural performance and affect the incidence of skeletal fractures.ConclusionsUnderstanding and preventing skeletal fractures is clinically important. Laboratory tests of whole bone strength are currently the only measures for in vivo fracture prediction. In the future, combined imaging and engineering models may be able to predict whole bone strength noninvasively.

Optical clearing in transcutaneous Raman spectroscopy of murine cortical bone tissue

The effect of optical clearing with glycerol on the Raman spectra of bone tissue acquired transcutaneously on right and left tibiae from four mice is studied. Multiple transcutaneous measurements are obtained from each limb; glycerol is then applied as an optical clearing agent, and additional transcutaneous measurements are taken. Glycerol reduces the noise in the raw spectra (p=0.0037) and significantly improves the cross-correlation between the recovered bone factor and the exposed bone measurement in a low signal-to-noise region of the bone spectra (p=0.0245).

Image-guided Raman spectroscopic recovery of canine cortical bone contrast in situ

Raman scattering provides valuable biochemical and molecular markers for studying bone tissue composition with use in predicting fracture risk in osteoporosis. Raman tomography can image through a few centimeters of tissue but is limited by low spatial resolution. X-ray computed tomography (CT) imaging can provide high-resolution image-guidance of the Raman spectroscopic characterization, which enhances the quantitative recovery of the Raman signals, and this technique provides additional information to standard imaging methods. This hypothesis was tested in data measured from Teflon tissue phantoms and from a canine limb. Image-guided Raman spectroscopy (IG-RS) of the canine limb using CT images of the tissue to guide the recovery recovered a contrast of 145:1 between the cortical bone and background. Considerably less contrast was found without the CT image to guide recovery. This study presents the first known IG-RS results from tissue and indicates that intrinsically high contrasts (on the order of a hundred fold) are available.

Fan-beam densitometry of the growing skeleton: are we measuring what we think we are?

Magnification error in fan-beam densitometers varies with distance from the X-ray source to the bone measured and might obscure bone mineral changes in the growing skeleton. Magnification was examined by scanning aluminum rods of different shapes (square, rectangular, solid round, and hollow round) at four distances above the X-ray source in two orientations, with rods aligned parallel (SI) and perpendicular (ML) to the longitudinal axis of the scanning table. Measured area (cm(2)) decreased linearly with distance above the X-ray source for all rods in the SI orientation (p < 0.005). Measured mineral content (g) decreased linearly with distance but only for SI round rods (p < 0.0001) and for ML hollow round rods (p < 0.005). Area and mineral content decreased 1.6-1.8% per centimeter above the source for round rods. Measured mineral density (g/cm(2)) decreased linearly with distance from the source only for ML hollow round rods (p < 0.005). Variation in area, mineral content, and mineral density measurements was 6.6-6.9%, 6.9-7.5%, and 1.9-2.3%, respectively, for SI round rods. Magnification errors of this magnitude are problematic for clinical studies using fan-beam densitometry. Particularly in pediatric subjects, increases in soft tissue during normal growth could increase a bones distance from the fan-beam source and result in apparent reductions in area and bone mineral content.

Correcting Fan-Beam Magnification in Clinical Densitometry Scans of Growing Subjects

As children grow, body and limb girths increase. For serial densitometric measurements, growth increases the distance between the bone region of interest and X-ray source over time, thereby increasing fan-beam magnification. To isolate bone accrual from magnification error in growing subjects, we developed a correction method based on waist girth, a common anthropometric measure. This correction was applied to dual-energy X-ray absorptiometry output obtained in a cohort of premenarcheal gymnasts and nongymnasts. After correcting for magnification, results for projected area and bone mineral content (BMC) increased by 0.4-1.1% at the lumbar spine and 8-16% at the femoral neck, decreasing areal bone mineral density (aBMD) by 0.4-2.3% at both sites. The effects of magnification correction were similar in magnitude to BMC and aBMD gains previously reported in longitudinal studies of normoactive children. Because of body size differences, the effect of correction for BMC and aBMD was 10-20% greater in nongymnasts than in gymnasts, which increased the observed aBMD differential between gymnasts and nongymnasts. Fan-beam magnification distorts true changes in bone mineral measures in growing premenarcheal girls and, therefore, may obscure additional activity-related changes during growth. Our correction technique may enhance detection of skeletal adaptation, particularly in pediatric populations.

Raman tomography of tissue phantoms and bone tissue

We report tomographic reconstruction of objects located several millimeters below the surface of highly scattering media. For this purpose we adapted proven software developed for fluorescence tomography with and without the use of spatial priors1. For this first demonstration we acquired Raman spectra using an existing ring/disk fiber optic probe with fifty collection fibers2. Several illumination ring diameters were employed to generate multiple angles of incidence. Tomographic reconstruction from Raman scatter was tested using a 9.5 mm diameter Teflon® sphere embedded in a gel of agarose and 1% Intralipid. Blind reconstruction of the sphere using the 732 cm-1 C-F stretch yielded an accurate shape but an inaccurate depth. Using the known shape and position of the object as spatial priors, a more accurate reconstruction was obtained. We also demonstrated a reconstruction of the tibial diaphysis of an intact canine hind limb using spatial priors generated from micro-computed tomography. In this first demonstration of Raman tomography in animal tissue, the P-O stretch of the bone mineral at 958 cm-1 was used for the reconstruction. An accurate shape and depth were recovered.

Biomechanics of Bone

The ability to bear loads is a critical function of the skeleton, in addition to its metabolic and physiological roles. Load-bearing ability depends on both the applied loads and the structural properties of the loaded bone. When the loads exceed the structural properties, fracture will occur. Because the nature of the applied loads can be difficult to predict, the greatest potential impact on minimizing fracture risk is through targeted interventions and therapies to improve bone strength. The strength and fracture resistance of the skeleton depend primarily on the mass, morphology/architecture, and material properties of bone tissue. Although each of these attributes has been examined independently in both cortical and cancellous bone, no single measurement can fully characterize the structural integrity of bone or reliably predict the occurrence of a fracture. In addition, factors such as aging, trauma, and disease affect the tissue properties and can compromise bone strength. While bone mass and, more recently, morphology have been widely examined in vivo, to date these measures do not fully explain variations in bone mechanical properties observed experimentally in vitro. Healthy bone tissue exhibits spatial and temporal variations in tissue-level material properties that are altered by aging and disease. Characterizing bone material properties, whether at the tissue level or at the chemical composition level of the mineral and matrix constituents, may improve the ability to predict structural competence and fracture risk reliably.

Mechanical and Vascular Cues Synergistically Enhance Osteogenesis in Human Mesenchymal Stem Cells.

Development and maintenance of a vascular network are critical for bone growth and homeostasis; strategies that promote vascular function are critical for clinical success of tissue-engineered bone constructs. Co-culture of endothelial cells (ECs) with mesenchymal stem cells (MSCs) and exposure to 10% cyclic tensile strain have both been shown to regulate osteogenesis in isolation, but potential synergistic effects have yet to be explored. The objective of this study was to expose an MSC-EC co-culture to 10% cyclic tensile strain to examine the role of this mechanical stimulus on MSC-EC behavior. We hypothesized that paracrine signaling from ECs would stimulate osteogenesis of MSCs, and exposure to 10% cyclic tensile strain would enhance this anabolic signal. Human umbilical vein ECs and human bone marrow-derived MSCs were either monocultured or co-cultured at a 1:1 ratio in a mixed osteo/angiogenic medium, exposed to 10% cyclic tensile strain at 1 Hz for 4 h/day for 2 weeks, and biochemically and histologically analyzed for endothelial and osteogenic markers. While neither 10% cyclic tensile strain nor co-culture alone had a significant effect on osteogenesis, the concurrent application of strain to an MSC-EC co-culture resulted in a significant increase in calcium accretion and mineral deposition, suggesting that co-culture and strain synergistically enhance osteogenesis. Neither co-culture, 10% cyclic tensile strain, nor a combination of these stimuli affected endothelial markers, indicating that the endothelial phenotype remained stable, but unresponsive to the stimuli evaluated in this study. This study is the first to investigate the role of cyclic tensile strain on the complex interplay between ECs and MSCs in co-culture. The results of this study provide key insights into the synergistic effects of 10% cyclic tensile strain and co-culture on osteogenesis. Understanding mechanobiological factors affecting MSC-EC crosstalk will help enhance strategies for creating vascularized tissues in tissue engineering and regenerative medicine.