Ryan K. Roeder

Gold nanoparticles as contrast agents in x-ray imaging and computed tomography

Computed tomography enables 3D anatomic imaging at a high spatial resolution, but requires delivery of an x-ray contrast agent to distinguish tissues with similar or low x-ray attenuation. Gold nanoparticles (AuNPs) have gained recent attention as an x-ray contrast agent due to exhibiting a high x-ray attenuation, nontoxicity and facile synthesis and surface functionalization for colloidal stability and targeted delivery. Potential diagnostic applications include blood pool imaging, passive targeting and active targeting, where actively targeted AuNPs could enable molecular imaging by computed tomography. This article summarizes the current state of knowledge for AuNP x-ray contrast agents within a paradigm of key structure-property-function relationships in order to provide guidance for the design of AuNP contrast agents to meet the necessary functional requirements in a particular application. Functional requirements include delivery to the site of interest (e.g., blood, tumors or microcalcifications), nontoxicity during delivery and clearance, targeting or localization at the site of interest and contrast enhancement for the site of interest compared with surrounding tissues. Design is achieved by strategically controlling structural characteristics (composition, mass concentration, size, shape and surface functionalization) for optimized properties and functional performance. Examples from the literature are used to highlight current design trade-offs that exist between the different functional requirements.

Osteoblast Responses One Hour After Load-Induced Fluid Flow in a Three-Dimensional Porous Matrix

When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres ~3 μm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.

Variability in Skeletal Mass, Structure, and Biomechanical Properties Among Inbred Strains of Rats

The aim of this study was to assess the usefulness of the inbred rat model for studies of genetic influences on skeletal fragility. We characterized bone mass, geometry, and skeletal biomechanics in 11 inbred strains of rats. This study showed that considerable variation exists in bone structure, areal bone mineral density (aBMD), and fragility phenotypes among inbred strains of rats. Interestingly, the variability in skeletal phenotypes in rats was site specific, suggesting that no single gene regulates skeletal fragility at all sites. For instance, the Copenhagen 2331 (COP) strain had the greatest biomechanical properties in the femoral neck but only modest bone strength at the femoral midshaft, compared with other strains. Consequently, COP rats appear to have alleles that specifically enhance femoral neck biomechanical properties and may serve as a model for studying genetic influences on hip strength. The Brown Norway (BN) and Fischer 344 (F344) strains may provide models for vertebral fragility because each has relatively fragile lumbar vertebrae. The F344 rats also had the most fragile femora and, thus, appear to carry alleles that cause overall skeletal fragility. We identified two inbred rat crosses that will facilitate the study of genetic influences on skeletal fragility at clinically relevant skeletal sites: Lewis (LEW) with F344 (primarily for vertebral fragility) and COP with DA (primarily for femoral neck fragility). The results strongly suggest that selected crosses of inbred strains of rats will provide useful models for studying genetic influences on bone strength and structure.

Hydroxyapatite reinforced collagen scaffolds with improved architecture and mechanical properties.

Hydroxyapatite (HA) reinforced collagen scaffolds have shown promise for synthetic bone graft substitutes and tissue engineering scaffolds. Freeze-dried HA-collagen scaffolds are readily fabricated and have exhibited osteogenicity in vivo, but are limited by an inherent scaffold architecture that results in a relatively small pore size and weak mechanical properties. In order to overcome these limitations, HA-collagen scaffolds were prepared by compression molding HA reinforcements and paraffin microspheres within a suspension of concentrated collagen fibrils (∼ 180 mg/mL), cross-linking the collagen matrix, and leaching the paraffin porogen. HA-collagen scaffolds exhibited an architecture with high porosity (85-90%), interconnected pores ∼ 300-400 μm in size, and struts ∼ 3-100 μm in thickness containing 0-80 vol% HA whisker or powder reinforcements. HA reinforcement enabled a compressive modulus of up to ∼ 1 MPa, which was an order of magnitude greater than unreinforced collagen scaffolds. The compressive modulus was also at least one order of magnitude greater than comparable freeze-dried HA-collagen scaffolds and two orders of magnitude greater than absorbable collagen sponges used clinically. Moreover, scaffolds reinforced with up to 60 vol% HA exhibited fully recoverable elastic deformation upon loading to 50% compressive strain for at least 100,000 cycles. Thus, the scaffold mechanical properties were well-suited for surgical handling, fixation, and bearing osteogenic loads during bone regeneration. The scaffold architecture, permeability, and composition were shown to be conducive to the infiltration and differentiation of adipose-derive stromal cells in vitro. Acellular scaffolds were demonstrated to induce angiogenesis and osteogenesis after subcutaneous ectopic implantation by recruiting endogenous cell populations, suggesting that the scaffolds were osteoinductive.

Anatomic variation in the elastic anisotropy of cortical bone tissue in the human femur.

Experimental investigations for anatomic variation in the magnitude and anisotropy of elastic constants in human femoral cortical bone tissue have typically focused on a limited number of convenient sites near the mid-diaphysis. However, the proximal and distal ends of the diaphysis are more clinically relevant to common orthopaedic procedures and interesting mechanobiology. Therefore, the objective of this study was to measure anatomic variation in the elastic anisotropy and inhomogeneity of human cortical bone tissue along the entire length (15%-85% of the total femur length), and around the periphery (anterior, medial, posterior and lateral quadrants) of the femoral diaphysis, using ultrasonic wave propagation in the three orthogonal specimen axes. The elastic symmetry of tissue in the distal and extreme proximal portions of the diaphysis (15%-45% and 75%-85% of the total femur length, respectively) was, at most, orthotropic. In contrast, the elastic symmetry of tissue near the mid- and proximal mid-diaphysis (50%-70% of the total femur length) was reasonably approximated as transversely isotropic. The magnitudes of elastic constants generally reached maxima near the mid- and proximal mid-diaphysis in the lateral and medial quadrants, and decreased toward the epiphyses, as well as the posterior and anterior quadrants. The elastic anisotropy ratio in the longitudinal and radial anatomic axes showed the opposite trends. These variations were significantly correlated with the apparent tissue density, as expected. In summary, the human femur exhibited statistically significant anatomic variation in elastic anisotropy, which may have important implications for whole bone numerical models and mechanobiology.

Hydroxyapatite whisker-reinforced polyetherketoneketone bone ingrowth scaffolds

Hydroxyapatite (HA) whisker-reinforced polyetherketoneketone (PEKK) bone ingrowth scaffolds were prepared and characterized. High levels of porosity (75-90%) and HA whisker reinforcement (0-40 vol.%) were attained using a powder processing approach to mix the HA whiskers, PEKK powder and a NaCl porogen, followed by compression molding at 350-375 degrees Celsius and particle leaching to remove the porogen. The scaffold architecture and microstructure exhibited characteristics known to be favorable for osteointegration. Scaffold porosity was interconnected with a mean pore size in the range 200-300 microm as measured by micro-computed tomography. HA whiskers were embedded within and exposed on the surface of scaffold struts, producing a microscale surface topography, shown by von Kossa staining and scanning electron microscopy. Therefore, HA whisker-reinforced PEKK bone ingrowth scaffolds may be advantageous for orthopedic implant fixation, including interbody spinal fusion.

Micro-computed tomography of fatigue microdamage in cortical bone using a barium sulfate contrast agent

Accumulation of microdamage during fatigue can lead to increased fracture susceptibility in bone. Current techniques for imaging microdamage in bone are inherently destructive and two-dimensional. Therefore, the objective of this study was to image the accumulation of fatigue microdamage in cortical bone using micro-computed tomography (micro-CT) with a barium sulfate (BaSO(4)) contrast agent. Two symmetric notches were machined on the tensile surface of bovine cortical bone beams in order to generate damage ahead of the stress concentrations during four-point bending fatigue. Specimens were loaded to a specified number of cycles or until one notch fractured, such that the other notch exhibited the accumulation of microdamage prior to fracture. Microdamage ahead of the notch was stained in vitro by precipitation of BaSO(4) and imaged using micro-CT. Reconstructed images showed a distinct region of bright voxels around the notch tip or along propagating cracks due to the presence of BaSO(4), which was verified by backscattered electron imaging and energy dispersive spectroscopy. The shape of the stained region ahead of the notch tip was consistent with principal strain contours calculated by finite element analysis. The relative volume of the stained region was correlated with the number of loading cycles by non-linear regression using a power-law. This study demonstrates new methods for the non-destructive and three-dimensional detection of fatigue microdamage accumulation in cortical bone in vitro, which may be useful to gain further understanding into the role of microdamage in bone fragility.

Mechanical properties of hydroxyapatite whisker reinforced polyetherketoneketone composite scaffolds

The apparent mechanical properties of hydroxyapatite (HA) whisker reinforced polyetherketoneketone (PEKK) scaffolds were evaluated in unconfined, uniaxial compression to investigate the effects of the porosity (75%, 82.5% and 90%), HA content (0, 20 and 40 vol%) and mold temperature (350, 365 and 375 ( composite function)C). Increased porosity resulted in a non-linear decrease in the elastic modulus and yield strength for both reinforced and unreinforced PEKK scaffolds, as expected. The increase in elastic modulus and yield strength with increased relative density followed a power-law, similar to trabecular bone and other open-cell foams. HA whisker reinforcement generally resulted in an increased elastic modulus from 0 to 20 vol% HA and a subsequent decrease from 20 to 40 vol% HA, while the yield strength and strain were decreased in scaffolds with 40 vol% HA compared to those with 0 or 20 vol% HA. Increased mold temperature resulted in an increased elastic modulus, yield strength and yield strain. These effects enabled the mechanical properties to be tailored to mimic human trabecular bone. The elastic modulus was greater than 50 MPa, and the yield strength was greater than 0.5 MPa, for scaffolds with 75% porosity at all combinations of reinforcement level and mold temperature. Scaffolds with 75% porosity and 20 vol% HA molded at 375 ( composite function)C exhibited a mean elastic modulus and yield strength of 149 MPa and 2.2 MPa, respectively, which was the highest of the conditions investigated in this study and similar to human vertebral trabecular bone. Therefore, HA whisker reinforced PEKK scaffolds may be advantageous for permanent implant fixation, including interbody spinal fusion.

Effects of the reinforcement morphology on the fatigue properties of hydroxyapatite reinforced polymers.

The objective of this study was to examine the effects of the hydroxyapatite (HA) reinforcement morphology and content on the fatigue behavior of HA reinforced high density polyethylene (HDPE). To this end, HDPE was reinforced with 20 and 40 vol% of either HA whiskers or an equiaxed HA powder, and tested in four-point bending fatigue under simulated physiological conditions. The fatigue life, mechanical property degradation and failure surfaces were compared between experimental groups. HDPE reinforced with HA whiskers exhibited a four- to five-fold increase (p < 0.001, T-test) in fatigue life compared to an equiaxed powder for either the 20 and 40 vol% reinforcement level. Composites containing 40 vol% HA exhibited decreased fatigue life compared to those with 20 vol% HA for either reinforcement morphology (p < 0.0001, ANOVA). HA whisker reinforced HDPE exhibited less stiffness loss, permanent deformation (creep) and energy dissipation at a given number of cycles compared to HA powder. Thus, HA whisker reinforced HDPE was more tolerant of fatigue damage due to either microcracking or polymer plasticity. Scanning electron microscopy of failure surfaces and surface microcracks showed evidence of toughening by uncracked ligaments, crack tip plasticity, polymer fibril bridging and HA whisker pullout. The results of this study suggest that the use of HA whiskers, in place of HA powder, is a straightforward means to improve the fatigue life and damage tolerance of HA reinforced polymers for synthetic bone substitutes.

Targeted delivery to bone and mineral deposits using bisphosphonate ligands.

The high concentration of mineral present in bone and pathological calcifications is unique compared with all other tissues and thus provides opportunity for targeted delivery of pharmaceutical drugs, including radiosensitizers and imaging probes. Targeted delivery enables accumulation of a high local dose of a therapeutic or imaging contrast agent to diseased bone or pathological calcifications. Bisphosphonates (BPs) are the most widely utilized bone-targeting ligand due to exhibiting high binding affinity to hydroxyapatite mineral. BPs can be conjugated to an agent that would otherwise have little or no affinity for the sites of interest. This article summarizes the current state of knowledge and practice for the use of BPs as ligands for targeted delivery to bone and mineral deposits. The clinical history of BPs is briefly summarized to emphasize the success of these molecules as therapeutics for metabolic bone diseases. Mechanisms of binding and the relative binding affinity of various BPs to bone mineral are introduced, including common methods for measuring binding affinity in vitro and in vivo. Current research is highlighted for the use of BP ligands for targeted delivery of BP conjugates in various applications, including (1) therapeutic drug delivery for metabolic bone diseases, bone cancer, other bone diseases, and engineered drug delivery platforms; (2) imaging probes for scintigraphy, fluorescence, positron emission tomography, magnetic resonance imaging, and computed tomography; and (3) radiotherapy. Last, and perhaps most importantly, key structure-function relationships are considered for the design of drugs with BP ligands, including the tether length between the BP and drug, the size of the drug, the number of BP ligands per drug, cleavable tethers between the BP and drug, and conjugation schemes.