John E. Novotny

In situ measurement of transport between subchondral bone and articular cartilage

Subchondral bone and articular cartilage play complementary roles in load bearing of the joints. Although the biomechanical coupling between subchondral bone and articular cartilage is well established, it remains unclear whether direct biochemical communication exists between them. Previously, the calcified cartilage between these two compartments was generally believed to be impermeable to transport of solutes and gases. However, recent studies found that small molecules could penetrate into the calcified cartilage from the subchondral bone. To quantify the real‐time solute transport across the calcified cartilage, we developed a novel imaging method based on fluorescence loss induced by photobleaching (FLIP). Diffusivity of sodium fluorescein (376 Da) was quantified to be 0.07 ± 0.03 and 0.26 ± 0.22 µm2/s between subchondral bone and calcified cartilage and within the calcified cartilage in the murine distal femur, respectively. Electron microscopy revealed that calcified cartilage matrix contained nonmineralized regions (∼22% volume fraction) that are either large patches (53 ± 18 nm) among the mineral deposits or numerous small regions (4.5 ± 0.8 nm) within the mineral deposits, which may serve as transport pathways. These results suggest that there exists a possible direct signaling between subchondral bone and articular cartilage, and they form a functional unit with both mechanical and biochemical interactions, which may play a role in the maintenance and degeneration of the joint.

Modeling fluorescence recovery after photobleaching in loaded bone: potential applications in measuring fluid and solute transport in the osteocytic lacunar-canalicular system.

Solute transport through the bone lacunar-canalicular system is essential for osteocyte viability and function, and it can be measured using fluorescence recovery after photobleaching (FRAP). The mathematical model developed here aims to analyze solute transport during FRAP in mechanically loaded bone. Combining both whole bone-level poroelasticity and cellular-level solute transport, we found that load-induced solute transport during FRAP is characterized by an exponential recovery rate, which is determined by the dimensionless Strouhal (St) number that characterizes the oscillation effects over the mean flows, and that significant transport occurs only for St values below a threshold, when the solute stroke displacement exceeds the distance between the source and sink (the canalicular length). This threshold mechanism explains the general flow behaviors such as increasing transport with increasing magnitude and decreasing frequency. Mechanical loading is predicted to enhance transport of all tracers relative to diffusion, with the greatest enhancement for medium-sized tracers and less enhancement for small and large tracers. This study provides guidelines for future FRAP experiments, based on which the model can be used to quantify bone permeability, solute–matrix interaction, and flow velocities. These studies should provide insights into bone adaptation and metabolism, and help to treat various bone diseases and conditions.

Cine phase contrast MRI to measure continuum Lagrangian finite strain fields in contracting skeletal muscle.

To measure the complex mechanics and Lagrangian finite strain of contracting human skeletal muscle in vivo with cine phase contrast MRI (CPC‐MRI) applied to the human supraspinatus muscle of the shoulder.

Anatomic Variations of the Lacunar-Canalicular System Influence Solute Transport in Bone

Solute transport in the lacunar-canalicular system (LCS) is essential for bone metabolism and mechanotransduction. Using the technique of fluorescence recovery after photobleaching (FRAP) we have been quantifying solute transport in the LCS of murine long bone as a function of loading parameters and molecular size. However, the influence of LCS anatomy, which varies among animal species, bone type and location, age and health condition, is not well understood. In this study, we developed a mathematical model to simulate solute convection in the LCS during a FRAP experiment under a physiological cyclic flow. We found that the transport rate (the reciprocal time constant for refilling the photobleached lacuna) increased linearly with canalicular number and decreased with canalicular length for both diffusion and convection. As a result, the transport enhancement of convection over diffusion was much less sensitive to the variations associated with chick, mouse, rabbit, bovine, dog, horse, and human LCS anatomy, when compared with the rates of diffusion or convection alone. Canalicular density did not affect transport enhancement, while solute size and the lacunar density had more complicated, non-linear effects. This parametric study suggests that solute transport could be altered by varying LCS parameters, and that the anatomical details of the LCS need systemic examination to further understand the etiology of aged and osteoporotic bones.

Effects of Hydrostatic Loading on a Self-Aggregating, Suspension Culture–Derived Cartilage Tissue Analog

Objective: Many approaches are being taken to generate cartilage replacement materials. The goal of this study was to use a self-aggregating suspension culture model of chondrocytes with mechanical preconditioning. Design: Our model differs from others in that it is based on a scaffold-less, self-aggregating culture model that produces a cartilage tissue analog that has been shown to share many similarities with the natural cartilage phenotype. Owing to the known loaded environment under which chondrocytes function in vivo, we hypothesized that applying force to the suspension culture–derived chondrocyte biomass would improve its cartilage-like characteristics and provide a new model for engineering cartilage tissue analogs. Results: In this study, we used a specialized hydrostatic pressure bioreactor system to apply mechanical forces during the growth phase to improve biochemical and biophysical properties of the biomaterial formed. We demonstrated that using this high-density suspension culture, a biomaterial more consistent with the hyaline cartilage phenotype was produced without any foreign material added. Unpassaged chondrocytes responded to a physiologically relevant hydrostatic load by significantly increasing gene expression of critical cartilage molecule collagen and aggrecan along with other cartilage relevant genes, CD44, perlecan, decorin, COMP, and iNOS. Conclusions: This study describes a self-aggregating bioreactor model without foreign material or scaffold in which chondrocytes form a cartilage tissue analog with many features similar to native cartilage. This study represents a promising scaffold-less, methodological advancement in cartilage tissue engineering with potential translational applications to cartilage repair.

An in-situ fluorescence-based optical extensometry system for imaging mechanically loaded bone

The application and quantification of well‐controlled tissue strains is required for investigations into mechanisms of tissue adaptation within the musculoskeletal system. Although many commercial and custom extensometry systems exist for large biological samples, integrated loading/strain measurement for small samples is not as readily available. Advanced imaging modules such as laser scanning microscopy provide in situ, minimally invasive tools to probe cellular and molecular processes with high spatiotemporal resolution. Currently, a need exists to devise loading/strain measurement systems that can be integrated with such advanced imaging modules. We describe the development and validation of a fluorescence‐based, optical extensometry system directly integrated within a confocal microscopy platform. This system allows in situ measurement of surface strain and is compatible with the direct imaging of cellular processes within small bone samples. This optical extensometry system can accurately and reproducibly measure physiologically relevant surface strains (200 to 3000 microstrain) in beams machined from various well‐characterized materials, including bovine femoral cortex, and in intact murine tibia. This simple system provides a powerful tool to further our investigation of the relationships between mechanical loading, fluid and solute transport, and mechanosensation within the musculoskeletal system.

Mechanical Characterization of a Nanotube-Polyethylene Composite Material

High-density polyethylene (HDPE) was used as the matrix material for a carbon nanotube (CNT) polymer composites. Multi-wall carbon nanotube composite films were fabricated using the melt processing method. Composite samples with 0%, 1%, 3% and 5% nanotube content by weight were tested. The mechanical properties of the films were measured by the small punch test and wear resistance was measured with a block-on-ring wear tester. Results show increases in the stiffness, peak load, work-to-failure and wear resistance with increasing nanotube content.© 2003 ASME

Maximum contractile strain in the biceps brachii is bounded by sarcomere geometry

Skeletal muscles’ primary function is the application of force to its bony origins and insertions. There are various models of muscle function that generally assume a uniform behavior from origin to insertion during force generation even though the structure and activation is complex. Engineering strains within skeletal muscles, though, have been shown to be non-uniform [1]. We have developed methods to quantify Lagrangian finite strains using cine phase-contrast magnetic resonance imaging (CPCMRI) and post-processing algorithms [2] and have described them during cyclic motion in the supraspinatus and biceps brachii. Principal and maximum in-plane shear strains can be identified at the scale of millimeters throughout the contracting and elongating muscle.Copyright

Measuring Diffusion in Non-Sectioned Articular Cartilage: A FRAP Sensitivity Study

Measuring the diffusion of molecules within articular cartilage is essential in characterizing its behavior. Information about this important mechanism may be useful to understanding changes in cartilage during degeneration or osteoarthritis. One method used in quantifying diffusion is fluorescence recovery after photobleaching (FRAP). The FRAP technique has been used in previous studies for cartilage [1] and various tissues [2]. In FRAP, a small region of interest (ROI) is selected within the tissue and fluorescent molecules are bleached using a higher laser power than would be used for imaging. Immediately following the ROI bleaching, bleached molecules diffuse out of the ROI as unbleached molecules diffuse into it. The average intensity data within the ROI is collected as a function of time. This data is then fit to a diffusion model usually resulting in a calculation of the diffusion constant, D (μm2/second) [3].Copyright

Non Invasive Measurement of Blood Flow in the Human Lower Extremity

Stress fracture in lower extremities occurs commonly among military recruits and athletes during intensive physical training. This injury has a marked impact on the health of military personnel and imposes a significant financial burden [1]. Despites advances in stress fracture studies, its pathogeneses remains poorly understood and early diagnostic tools are lacking. Focal ischemia is a potential initiator of site-specific bone remodeling and may cause stress fractures in human lower extremity [2, 3]. We hypothesize that intensive repetitive loading impairs intramedullary blood flow due to pressurization of the bone marrow cavity, leading to focal ischemia and eventual development of stress fractures. To begin to test our hypothesis, we developed and validated a quantitative, non-invasive method to measure blood flow in vivo. The approach was based on Cine Phase Contrast MRI (CPC-MRI), a dynamic motion measurement and visualization modality that was originally designed for cardiovascular studies. By measuring the phase shift that is induced by pulsatile blood flows in the magnetic resonance signal, cross-sectional images and velocity maps are acquired of the moving fluid. This technique has been adapted to study blood flows in skeletal muscles [4], intracranial flows [5], and muscle mechanics and joint kinematics [6]]. We first performed a flow phantom study to validate the reliability and accuracy of CPC-MRI in measuring flow velocity. We then quantified the effects of brief exercise on blood flows in the lower extremities of human subjects. These non-invasive measurements will help us better understand the interplay between vasculature and skeletal system in various physiological and pathological conditions.Copyright