Marc E. Levenston

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.

A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants

We have developed an incubator housed, biaxial-tissue-loading device capable of applying axial deformations as small as 1 microm and sinusoidal rotations as small as 0.01 degrees. Axial resolution is 50 nm for applying sinewaves as low as 10 microm (or 1% based on a 1 mm thickness) or as large as 100 microm. Rotational resolution is 0.0005 degrees. The machine is small enough (30 cm high x 25 cm x 20 cm) to be placed in a standard incubator for long-term tissue culture loading studies. In metabolic studies described here, application of sinusoidal macroscopic shear deformation to articular cartilage explants resulted in a significant increase in the synthesis of proteoglycan and proteins (uptake of (35)S-sulfate and (3)H-proline) over controls held at the same static offset compression.

Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels

Articular cartilage responds to its mechanical environment through altered cell metabolism and matrix synthesis. In this study, isolated articular chondrocytes were cultured in collagen type I gels and exposed to uniaxial static compression of 0%, 25%, or 50% of original thickness for 0.5, 4, and 24 h, and to oscillatory (25 +/- 4%, 1 Hz) compression for 24 h. The cellular response was assessed through competitive and real-time RT-PCR to quantify expression of genes for collagen type I, collagen type II, and aggrecan core protein, and through radiolabelled proline and sulfate incorporation to quantify protein and proteoglycan synthesis rates. Static compression for 24 h inhibited expression of collagen I and II mRNAs and inhibited 3H-proline and 35S-sulfate incorporation. The mRNA expression exhibited transient fluctuations at intermediate time points. Oscillatory compression had no effect upon mRNA expression, and 24 h after release from static compression, there was no difference in collagen II or aggrecan mRNA, while there was an inhibition of collagen I. We conclude that the chondrocytes maintained some aspects of their ability to sense and respond to static compression, despite a biochemical and mechanical environment which is different from that in tissue. This suggests that mechanical stimuli may be useful in modulating chondrocyte metabolism in tissue engineering systems using fibrillar protein scaffolds.

Biomechanical analysis of silicon microelectrode-induced strain in the brain

The ability to successfully interface the brain to external electrical systems is important both for fundamental understanding of our nervous system and for the development of neuroprosthetics. Silicon microelectrode arrays offer great promise in realizing this potential. However, when they are implanted into the brain, recording sensitivity is lost due to inflammation and astroglial scarring around the electrode. The inflammation and astroglial scar are thought to result from acute injury during electrode insertion as well as chronic injury caused by micromotion around the implanted electrode. To evaluate the validity of this assumption, the finite element method (FEM) was employed to analyze the strain fields around a single Michigan Si microelectrode due to simulated micromotion. Micromotion was mimicked by applying a force to the electrode, fixing the boundaries of the brain region and applying appropriate symmetry conditions to nodes lying on symmetry planes. Characteristics of the deformation fields around the electrode including maximum electrode displacement, strain fields and relative displacement between the electrode and the adjacent tissue were examined for varying degrees of physical coupling between the brain and the electrode. Our analysis demonstrates that when physical coupling between the electrode and the brain increases, the micromotion-induced strain of tissue around the electrode decreases as does the relative slip between the electrode and the brain. These results support the use of neuro-integrative coatings on electrode arrays as a means to reduce the micromotion-induced injury response.

Dynamic Compression Regulates the Expression and Synthesis of Chondrocyte-Specific Matrix Molecules in Bone Marrow Stromal Cells

The overall objective of the present study was to investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor β1 (TGF‐β1), dexamethasone, and dynamic compressive loading. Overall, the addition of TGF‐β1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. In contrast, there was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF‐β1 and dexamethasone being the most responsive at day 16. Both collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF‐β1 media. These findings suggest that interactions between mechanical stimuli and TGF‐β signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis.

Quantitative Assessment of Articular Cartilage Morphology via EPIC-μCT

OBJECTIVE The objective of the present study was to validate the ability of Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-microCT) to nondestructively assess cartilage morphology in the rat model. DESIGN An appropriate contrast agent (Hexabrix) concentration and incubation time for equilibration were determined for reproducible segmentation of femoral articular cartilage from contrast-enhanced microCT scans. Reproducibility was evaluated by triplicate scans of six femora, and the measured articular cartilage thickness was independently compared to thickness determined from needle probe testing and histology. The validated technique was then applied to quantify age-related differences in articular cartilage morphology between 4, 8, and 16-week-old (n=5 each) male Wistar rats. RESULTS A 40% Hexabrix/60% phosphate buffered saline (PBS) solution with 30 min incubation was optimal for segmenting cartilage from the underlying bone tissue and other soft tissues in the rat model. High reproducibility was indicated by the low coefficient of variation (1.7-2.5%) in cartilage volume, thickness and surface area. EPIC-microCT evaluation of thickness showed a strong linear relationship and good agreement with both needle probing (r(2)=0.95, slope=0.81, P<0.01, mean difference 11+/-22 microm, n=43) and histology (r(2)=0.99, slope=0.97, P<0.01, mean difference 12+/-10 microm, n=30). Cartilage volume and thickness significantly decreased with age while surface area significantly increased. CONCLUSION EPIC-microCT imaging has the ability to nondestructively evaluate three-dimensional articular cartilage morphology with high precision and accuracy in a small animal model.

3D imaging of tissue integration with porous biomaterials

Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.

Interactions between integrin ligand density and cytoskeletal integrity regulate BMSC chondrogenesis

Interactions with the extracellular matrix play important roles in regulating the phenotype and activity of differentiated articular chondrocytes; however, the influences of integrin‐mediated adhesion on the chondrogenesis of mesenchymal progenitors remain unclear. In the present study, agarose hydrogels were modified with synthetic peptides containing the arginine‐glycine‐aspartic acid (RGD) motif to investigate the effects of integrin‐mediated adhesion and cytoskeletal organization on the chondrogenesis of bone marrow stromal cells (BMSCs) within a three‐dimensional culture environment. Interactions with the RGD‐modified hydrogels promoted BMSC spreading in a density‐dependent manner and involved αvβ3 integrin receptors. When cultured with the chondrogenic supplements, TGF‐β1 and dexamethasone, adhesion to the RGD sequence inhibited the stimulation of sulfated‐glycosaminoglycan (sGAG) production in a RGD density‐dependent manner, and this inhibition could be blocked by disrupting the F‐actin cytoskeleton with cytochalasin D. In addition, interactions with the RGD‐modified gels promoted cell migration and aggrecanase‐mediated release of sGAG to the media. While adhesion to the RGD sequence inhibited BMSC chondrogenesis in the presence of TGF‐β1 and dexamethasone, osteocalcin and collagen I gene expression and alkaline phosphatase activity were enhanced by RGD interactions in the presence of serum‐supplemented medium. Overall, the results of this study demonstrate that integrin‐mediated adhesion within a three‐dimensional environment inhibits BMSC chondrogenesis through actin cytoskeleton interactions. Furthermore, the effects of RGD‐adhesion on mesenchymal differentiation are lineage‐specific and depend on the biochemical composition of the cellular microenvironment. J. Cell. Physiol. 217: 145–154, 2008.

Nondestructive Assessment of sGAG Content and Distribution in Normal and Degraded Rat Articular Cartilage via EPIC-μCT

OBJECTIVE The objective of this study was to evaluate the feasibility of quantifying the Equilibrium Partitioning of an Ionic Contrast agent via Microcomputed Tomography (EPIC-microCT) to nondestructively assess sulfated glycosaminoglycan (sGAG) content and distribution in rat articular cartilage ex vivo, and in doing so to establish a paradigm for extension of this technique to other small animal models. DESIGN After determination of an appropriate incubation time for the anionic contrast agent, EPIC-microCT was used to examine age-related differences in cartilage sGAG content between 4-, 8-, and 16-week old (n=5 each) male Wistar rats and to evaluate sGAG depletion in the right femora of each age group after 60 min of digestion with chondroitinase ABC. The EPIC-microCT measurements were validated by histological safranin-O staining, and reproducibility was evaluated by triplicate scans of six femora. RESULTS Cartilage attenuation gradually increased with cumulative digestion time and reached a plateau at approximately 60 min with a 16.0% temporal increase (P<0.01). Average femoral articular cartilage attenuation increased by 14.2% from 4- to 8-weeks of age (P<0.01) and further increased by 2.5% from 8 to 16 weeks (P<0.05). After 60 min of digestion, femoral articular cartilage attenuations increased by 15-17% in each age group (P<0.01). Correspondingly, sGAG optical density decreased with age and digestion, and showed a linear correlation (r=-0.88, slope=-1.26, P<0.01, n=30) with EPIC-microCT cartilage attenuation. High reproducibility was indicated by a low coefficient of variation (1.5%) in cartilage attenuation. CONCLUSIONS EPIC-microCT imaging provides high spatial resolution and sensitivity to assess sGAG content and three-dimensional distribution in rat femoral articular cartilage.

Computer simulations of stress-related bone remodeling around noncemented acetabular components

The authors have used computer modeling techniques to examine stress-related bone changes in the acetabular region. Using a previously developed theory for bone development and adaptation, the authors simulated the distribution of bone density in the natural pelvis as well as changes in bone density following total hip arthroplasty. The geometry of the finite element model was based on a two-dimensional slice through the pelvis. Starting from a solid, homogeneous structure, the computer simulations predicted the distribution of bone density throughout the natural pelvis. The predicted bone density distribution in this first simulation agreed well with the actual bone density distribution only when loads representing multiple activities were incorporated. Using the predicted density distribution as a starting point the authors modified the finite element models to study two designs of noncemented, metal-backed acetabular cups. The simulations with fully fixed bone-implant interfaces predicted extensive loss of bone density medial and inferior to the prosthetic components. The simulations with loose interfaces led to more moderate losses of bone density, indicating a load transfer more similar to that which occurs in the natural joint. The differences in simulated bone remodeling between the two component designs were quite minimal. These results indicate that acetabular components with full bony ingrowth may induce significant stress-related bone remodeling due to a nonphysiologic transfer of load.