Robert E. Guldberg

Guidelines for assessment of bone microstructure in rodents using micro-computed tomography

Use of high‐resolution micro–computed tomography (µCT) imaging to assess trabecular and cortical bone morphology has grown immensely. There are several commercially available µCT systems, each with different approaches to image acquisition, evaluation, and reporting of outcomes. This lack of consistency makes it difficult to interpret reported results and to compare findings across different studies. This article addresses this critical need for standardized terminology and consistent reporting of parameters related to image acquisition and analysis, and key outcome assessments, particularly with respect to ex vivo analysis of rodent specimens. Thus the guidelines herein provide recommendations regarding (1) standardized terminology and units, (2) information to be included in describing the methods for a given experiment, and (3) a minimal set of outcome variables that should be reported. Whereas the specific research objective will determine the experimental design, these guidelines are intended to ensure accurate and consistent reporting of µCT‐derived bone morphometry and density measurements. In particular, the methods section for papers that present µCT‐based outcomes must include details of the following scan aspects: (1) image acquisition, including the scanning medium, X‐ray tube potential, and voxel size, as well as clear descriptions of the size and location of the volume of interest and the method used to delineate trabecular and cortical bone regions, and (2) image processing, including the algorithms used for image filtration and the approach used for image segmentation. Morphometric analyses should be based on 3D algorithms that do not rely on assumptions about the underlying structure whenever possible. When reporting µCT results, the minimal set of variables that should be used to describe trabecular bone morphometry includes bone volume fraction and trabecular number, thickness, and separation. The minimal set of variables that should be used to describe cortical bone morphometry includes total cross‐sectional area, cortical bone area, cortical bone area fraction, and cortical thickness. Other variables also may be appropriate depending on the research question and technical quality of the scan. Standard nomenclature, outlined in this article, should be followed for reporting of results.

The hypoxia-inducible factor α pathway couples angiogenesis to osteogenesis during skeletal development

Skeletal development and turnover occur in close spatial and temporal association with angiogenesis. Osteoblasts are ideally situated in bone to sense oxygen tension and respond to hypoxia by activating the hypoxia-inducible factor alpha (HIF alpha) pathway. Here we provide evidence that HIF alpha promotes angiogenesis and osteogenesis by elevating VEGF levels in osteoblasts. Mice overexpressing HIF alpha in osteoblasts through selective deletion of the von Hippel-Lindau gene (Vhl) expressed high levels of Vegf and developed extremely dense, heavily vascularized long bones. By contrast, mice lacking Hif1a in osteoblasts had the reverse skeletal phenotype of that of the Vhl mutants: long bones were significantly thinner and less vascularized than those of controls. Loss of Vhl in osteoblasts increased endothelial sprouting from the embryonic metatarsals in vitro but had little effect on osteoblast function in the absence of blood vessels. Mice lacking both Vhl and Hif1a had a bone phenotype intermediate between those of the single mutants, suggesting overlapping functions of HIFs in bone. These studies suggest that activation of the HIF alpha pathway in developing bone increases bone modeling events through cell-nonautonomous mechanisms to coordinate the timing, direction, and degree of new blood vessel formation in bone.

Mechanical properties of a novel PVA hydrogel in shear and unconfined compression

Poly(vinyl alcohol) (PVA) hydrogels have been proposed as promising biomaterials to replace diseased or damaged articular cartilage. A critical barrier to their use as load-bearing tissue replacements is a lack of sufficient mechanical properties. The purpose of this study was to characterize the functional compressive and shear mechanical properties of a novel PVA hydrogel. Two formulations of the biomaterial were tested, one with a lower water content (75% water), and the other with higher water content (80% water). The compressive tangent modulus varied with biomaterial formulation and was found to be statistically strain magnitude and rate dependent. Over a strain range of 10-60%, the compressive modulus increased from approximately 1-18 MPa, which is within the range of the modulus of articular cartilage. The shear tangent modulus (0.1-0.4 MPa) was also found to be strain magnitude dependent and within the range of normal human articular cartilage, but it was not statistically dependent on strain rate, This behavior was attributed to the dominance of fluid flow and related frictional drag on the viscoelastic behavior. Compressive failure of the hydrogels was found to occur between 45 and 60% strain, depending on water content.

Effects of Medium Perfusion Rate on Cell-Seeded Three-Dimensional Bone Constructs in Vitro

Cellular activity at the center of tissue-engineered constructs in static culture is typically decreased relative to the construct periphery because of transport limitations. We have designed a tissue culture system that perfuses culture medium through three-dimensional (3D) porous cellular constructs to improve nutrient delivery and waste removal within the constructs. This study examined the effects of medium perfusion rate on cell viability, proliferation, and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for 1 week at flow rates of 0.01, 0.1, 0.2, and 1.0 mL/min. Confocal microscopy after 1 week of culture indicated that a flow rate of 1.0 mL/min resulted in substantial cell death throughout the constructs whereas lowering the flow rate led to an increasing proportion of viable cells, particularly at the center of the constructs. DNA analysis showed increases in cell proliferation at a flow rate of 0.01 mL/min relative to 0.2 mL/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin, and alkaline phosphatase were upregulated at 0.2 mL/min compared with lower flow rates as quantified by real-time RT-PCR. These data suggest that medium perfusion may benefit the development of 3-D tissues in vitro by enhancing transport of nutrients and waste within the constructs and providing flow-mediated mechanical stimuli.

Microarchitectural and mechanical characterization of oriented porous polymer scaffolds.

Biodegradable porous polymer scaffolds are widely used in tissue engineering to provide a structural template for cell seeding and extracellular matrix formation. Scaffolds must often possess sufficient structural integrity to temporarily withstand functional loading in vivo or cell traction forces in vitro. Both the mechanical and biological properties of porous scaffolds are determined in part by the local microarchitecture. Quantification of scaffold structure-function relationships is therefore critical for optimizing mechanical and biological performance. In this study, porous poly(L-lactide-co-DL-lactide) scaffolds with axially oriented macroporosity and random microporosity were produced using a solution coating and porogen decomposition method. Microarchitectural parameters were quantified as a function of porogen concentration using microcomputed tomography (micro-CT) analysis and related to compressive mechanical properties. With increasing porogen concentration, volume fraction decreased consistently due to microarchitectural changes in average strut thickness, spacing, and density. The three-dimensional interconnectivity of the scaffold porosity was greater than 99% for all porogen concentration levels tested. Over a porosity range of 58-80%, the average compressive modulus and ultimate strength of the scaffolds ranged from 43.5-168.3 MPa and 2.7-11.0 MPa, respectively. Thus, biodegradable porous polymer scaffolds have been produced with oriented microarchitectural features designed to facilitate vascular invasion and cellular attachment and with initial mechanical properties comparable to those of trabecular bone.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal μ-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. μ-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Inactivation of the Osteopontin Gene Enhances Vascular Calcification of Matrix Gla Protein–deficient Mice: Evidence for Osteopontin as an Inducible Inhibitor of Vascular Calcification In Vivo

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP−/− OPN+/+) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 ± 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM α-actin and SM22α. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP−/− OPN+/+ at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 ± 0.2 wk) than MGP−/− OPN+/+ counterparts (6.6 ± 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.

Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP‐2‐producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene‐enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.

Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography

Small animal models of osteoarthritis are often used for evaluating the efficacy of pharmacologic treatments and cartilage repair strategies, but noninvasive techniques capable of monitoring matrix-level changes are limited by the joint size and the low radiopacity of soft tissues. Here we present a technique for the noninvasive imaging of cartilage at micrometer-level resolution based on detecting the equilibrium partitioning of an ionic contrast agent via microcomputed tomography. The approach exploits electrochemical interactions between the molecular charges present in the cartilage matrix and an ionic contrast agent, resulting in a nonuniform equilibrium partitioning of the ionic contrast agent reflecting the proteoglycan distribution. In an in vitro model of cartilage degeneration we observed changes in x-ray attenuation magnitude and distribution consistent with biochemical and histological analyses of sulfated glycosaminoglycans, and x-ray attenuation was found to be a strong predictor of sulfated glycosaminoglycan density. Equilibration with the contrast agent also permits direct in situ visualization and quantification of cartilage surface morphology. Equilibrium partitioning of an ionic contrast agent via microcomputed tomography thus provides a powerful approach to quantitatively assess 3D cartilage composition and morphology for studies of cartilage degradation and repair.

Hemodynamic Shear Stresses in Mouse Aortas: Implications for Atherogenesis

Objective—The hemodynamic environment is a determinant of susceptibility to atherosclerosis in the vasculature. Although mouse models are commonly used in atherosclerosis studies, little is known about local variations in wall shear stress (WSS) in the mouse and whether the levels of WSS are comparable to those in humans. The objective of this study was to determine WSS values in the mouse aorta and to relate these to expression of gene products associated with atherosclerosis. Methods and Results—Using micro-CT and ultrasound methodologies we developed a computational fluid dynamics model of the mouse aorta and found values of WSS to be much larger than those for humans. We also used a quantum dot-based approach to study vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression on the aortic intima and demonstrated that increased expression for these molecules occurs where WSS was relatively low for the mouse. Conclusions—Despite large differences in WSS in the two species, the spatial distributions of atherogenic molecules in the mouse aorta are similar to atherosclerotic plaque localization found in human aortas. These results suggest that relative differences in WSS or in the direction of WSS, as opposed to the absolute magnitude, may be relevant determinants of flow-mediated inflammatory responses.