The effects of loading on cancellous bone in the rabbit.

Mechanical stimuli are critical to the growth, maintenance, and repair of the skeleton. The adaptation of bone to mechanical forces has primarily been studied in cortical bone. As a result, the mechanisms of bone adaptation to mechanical forces are not well-understood in cancellous bone. Clinically, however, diseases such as osteoporosis primarily affect cancellous tissue and mechanical solutions could counteract cancellous bone loss. We previously developed an in vivo model in the rabbit to study cancellous functional adaptation by applying well-controlled mechanical loads to cancellous sites. In the rabbit, in vivo loading of the lateral aspect of the distal femoral condyle simulated the in vivo bone-implant environment and enhanced bone mass. Using animal-specific computational models and further in vivo experiments we demonstrate here that the number of loading cycles and loading duration modulate the cancellous response by increasing bone volume fraction and thickening trabeculae to reduce the strains experienced in the bone tissue with loading and stiffen the tissue in the loading direction.

Imaging of gene expression in live pancreatic islet cell lines using dual-isotope SPECT.

UNLABELLED: We are combining nuclear medicine with molecular biology to establish a sensitive, quantitative, and tomographic method with which to detect gene expression in pancreatic islet cells in vivo. Dual-isotope SPECT can be used to image multiple molecular events simultaneously, and coregistration of SPECT and CT images enables visualization of reporter gene expression in the correct anatomic context. We have engineered pancreatic islet cell lines for imaging with SPECT/CT after transplantation under the kidney capsule. METHODS: INS-1 832/13 and alphaTC1-6 cells were stably transfected with a herpes simplex virus type 1-thymidine kinase-green fluorescent protein (HSV1-thymidine kinase-GFP) fusion construct (tkgfp). After clonal selection, radiolabel uptake was determined by incubation with 5-(131)I-iodo-1-(2-deoxy-2-fluoro-beta-d-arabinofuranosyl)uracil ((131)I-FIAU) (alphaTC1-6 cells) or (123)I-FIAU (INS-1 832/13 cells). For the first set of in vivo experiments, SPECT was conducted after alphaTC1-6/tkgfp cells had been labeled with either (131)I-FIAU or (111)In-tropolone and transplanted under the left kidney capsule of CD1 mice. Reconstructed SPECT images were coregistered to CT. In a second study using simultaneous acquisition dual-isotope SPECT, INS-1 832/13 clone 9 cells were labeled with (111)In-tropolone before transplantation. Mice were then systemically administered (123)I-FIAU and data for both (131)I and (111)In were acquired simultaneously. RESULTS: alphaTC1-6/tkgfp cells showed a 15-fold greater uptake of (131)I-FIAU, and INS-1/tkgfp cells showed a 12-fold greater uptake of (123)I-FIAU, compared with that of wild-type cells. After transplantation under the kidney capsule, both reporter gene expression and location of cells could be visualized in vivo with dual-isotope SPECT. Immunohistochemistry confirmed the presence of glucagon- and insulin-positive cells at the site of transplantation. CONCLUSION: Dual-isotope SPECT is a promising method to detect gene expression in and location of transplanted pancreatic cells in vivo.

Optical imaging of hydroxyapatite in the calcified vasculature of transgenic animals.

OBJECTIVE: To detect the hydroxyapatite component of vascular calcification in vivo so that the process of calcium deposition can be studied in transgenic model systems. METHODS AND RESULTS: We have previously developed a near-infrared fluorescent bisphosphonate derivative that binds with high affinity and specificity to hydroxyapatite, and an intraoperative near-infrared fluorescence imaging system for small animals. Using these tools, and a transgenic mouse strain with homozygous deletion of the matrix GLA protein (Mgp(-/-)), we demonstrate that the hydroxyapatite component of vascular calcification can be detected in vivo with high sensitivity, specificity, and resolution. CONCLUSIONS: The hydroxyapatite component of vascular calcification can be detected optically, in real-time, without sacrifice of the animal. It is now possible to study the earliest events associated with vascular mineralization, at the cell and organ level, and to monitor the process in living animals.

Cancellous bone adaptation to in vivo loading in a rabbit model.

Biophysical stimuli are important to the development and maintenance of cancellous bone, but the regulatory mechanisms need to be understood. We investigated the effects of mechanical loading applied in vivo to native cancellous bone in the rabbit on bone formation and trabecular realignment. A novel device was developed to apply controlled compressive loads to cancellous bone in situ. The effect of loading on cancellous bone volume fraction and architecture was quantified. A 4-week experiment was performed in rabbits with devices implanted bilaterally. Cyclic 1 MPa pressures were applied daily to the right limb for 10, 25, or 50 cycles at 0.5 Hz, and the left limb served as the control without any applied loading. Microcomputed tomography and histomorphometry were used to characterize the cancellous tissue within a 4-mm spherical volume located below the loading core. In vivo cyclic loading significantly increased the bone volume fraction, direct trabecular thickness, mean intercept length, and mineral apposition rate in the loaded limbs compared with contralateral limbs. Insufficient evidence was found to demonstrate an effect of number of cycles on the cancellous adaptation between loaded and control limbs. Using a rabbit model, we demonstrated that mechanical loading applied to cancellous bone in situ increased bone formation and altered trabecular morphology. This in vivo model will allow further investigation of cancellous functional adaptation to controlled mechanical stimuli and the influence of mechanical loading parameters, metabolic status, and therapeutic agents.

Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons.

We demonstrate how to construct calibrated, stable, and inexpensive tissue-like phantoms for near-IR (NIR) fluorescence imaging applications. The bulk phantom material is composed of gelatin, intralipid, hemoglobin, and indocyanine green (ICG). Absorbance, scatter, background fluorescence, and texture can be tuned as desired. NIR fluorescent inclusions are comprised of ICG-labeled polystyrene divinylbenzene beads and Pam78-labeled hydroxyapatite crystals. The former mimic tumor masses of controllable size and contrast agent concentration, and the latter mimic microcalcifications in breast cancer. NIR-fluorescent inclusions can be positioned precisely in phantoms, with one or more regions having different optical properties, and their position can be verified independently using microcomputed tomography. We demonstrate how these phantoms can be used to calibrate and compare imaging systems, and to train surgeons to operate under NIR fluorescence image guidance.

Genetic variation in structure-function relationships for the inbred mouse lumbar vertebral body.

UNLABELLED: Structure-function relationships were determined for L(5) vertebral bodies from three inbred mouse strains. Genetic variability in whole bone mechanical properties could be explained by a combination of the traits specifying the amount, distribution, and quality of the cortical and trabecular bone tissue. INTRODUCTION: Although phenotypically correlated with fracture, BMD may be disadvantageous to use in genetic and biomechanical analyses because BMD does not distinguish the contributions of the underlying morphological and compositional bone traits. Developing functional relationships between the underlying bone traits and whole bone mechanical properties should further our understanding of the genetics of bone fragility. MATERIALS AND METHODS: Microarchitecture and composition of L(5) vertebral bodies (n = 10/strain) from A/J, C57BL/6J, and C3H/HeJ inbred mouse strains were determined using muCT with an isotropic voxel size of 16 mum(3). Failure load, stiffness, and total deformation as a measure of ductility were measured in compression using a noncontact strain extensometer imaging system. A correlation analysis related morphological and compositional bone traits to whole bone mechanical properties. A multivariate analysis identified structure-function relationships for each genotype. RESULTS: No single bone trait accurately explained the genetic variation in mechanical properties. However, a combination of traits describing the amount, distribution, and quality of cortical and trabecular bone tissue explained >70% of the variation in vertebral mechanical properties. Importantly, structure-function relationships were unique among genotypes. CONCLUSIONS: Different genetic backgrounds use different combinations of underlying bone traits to create mechanically functional structures. Using a single complex trait such as BMD or BV/TV as the sole phenotypic marker in genetic analyses may prove to be disadvantageous because of the complex relationship between mechanical properties and the underlying bone traits. Therefore, considering multiple bone traits and the interaction among these bone traits is necessary to understand the relationship between genetic background and complex whole bone mechanical properties.

Tissue-level remodeling simulations of cancellous bone capture effects of in vivo loading in a rabbit model.

The adaptation of cancellous bone to mechanical stimuli occurs throughout normal skeletal growth and aging, as well as in response to surgery, disease and device implantation. Previously we developed an in vivo cancellous loading model in the distal lateral femur of the rabbit. In response to daily in vivo loading for four weeks, bone mass increased, trabeculae thickened and the apparent modulus of the underlying cancellous bone increased. Here, we simulated our prior in vivo rabbit loading experiment using a cell-based tissue remodeling algorithm (Mullender et al., 1994) and compared the results to the in vivo experimental data published previously. Cancellous bone tissue was added or removed from the surface of trabeculae in regions of high and low mechanical stimulus, respectively. To examine the effect of material properties on mechanically regulated adaptation, we implemented both a homogeneous material model and a model where the relative density of tissue was lower for new and surface bone tissue compared to interior tissue. The simulations captured the changes in histomorphometric parameters and mechanical properties measured in the in vivo experiment illustrating the ability of computational simulations to predict the effect of mechanically regulated adaptation on cancellous bone histomorphometry and apparent modulus.

Carnosine disaggregates glycated alpha-crystallin: an in vitro study.

Protein glycation, which promotes aggregation, involves the unwanted reaction of carbohydrate oxidation products with proteins. Glycation of lens alpha-crystallin occurs in vivo and may contribute to cataractogenesis. Anti-glycation compounds such as carnosine may be preventive, but interestingly carnosine reverses lens opacity in human trials. The mechanism for this observation may involve carnosine's ability to disaggregate glycated protein. We investigated this hypothesis using glycated alpha-crystallin as our in vitro model. Methylglyoxal-induced glycation of alpha-crystallin caused aggregation as evidenced by increased 90 degrees light scattering. After addition of carnosine, light scattering returned to baseline levels suggesting that the size of the glycation-induced aggregates decreased. Additionally, carnosine decreased tryptophan fluorescence polarization of glycated alpha-crystallin, suggesting that carnosine increased peptide chain mobility, which may contribute to the controlled unfolding of glycated protein. Comparatively, guanidine-HCl and urea had no effect. Our data support the hypothesis that carnosine disaggregates glycated alpha-crystallin.