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.

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.

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 microm(2)/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 (approximately 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.

WEAR BEHAVIOR OF CARBON NANOTUBE/HIGH DENSITY POLYETHYLENE COMPOSITES.

Carbon Nanotube/High Density Polyethylene (CNT/HDPE) composites were manufactured and tested to determine their wear behavior. The nanocomposites were made from untreated multi-walled carbon nanotubes and HDPE pellets. Thin films of the precursor materials were created with varying weight percentages of nanotubes (1%, 3%, and 5%), through a process of mixing and extruding. The precursor composites were then molded and machined to create test specimens for mechanical and wear tests. These included small punch testing to compare stiffness, maximum load and work-to-failure and block-on-ring testing to determine wear behavior. Each of the tests was conducted for the different weight percentages of composite as well as pure HDPE as the baseline. The measured mechanical properties and wear resistance of the composite materials increased with increasing nanotube content in the range studied.

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.

PURPOSE: 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. MATERIALS AND METHODS: Processing techniques are applied to transform velocities from CPC-MRI images to displacements and planar Lagrangian finite strain. An interpolation method describing the continuity of the velocity field and forward-backward and Fourier transform methods were used to track the displacement of regions of interest during a cyclic abduction motion of a subject's arm. The components of the Lagrangian strain tensor were derived during the motion and principal and maximum in-plane shear strain fields calculated. RESULTS: Derived displacement and strain fields are shown that describe the contraction mechanics of the supraspinatus. Strains vary over time during the cyclic motion and are highly nonuniform throughout the muscle. CONCLUSION: This method presented overcomes the physical resolution of the MRI scanner, which is crucial for the detection of detailed information within muscles, such as the changes that might occur with partial tears of the supraspinatus. These can then be used as input or validation data for modeling human skeletal muscle.

Biomechanical and magnetic resonance characteristics of a cartilage-like equivalent generated in a suspension culture.

OBJECTIVE: To generate a cartilage biomaterial using a suspension culture with biophysical properties similar to native articular cartilage. DESIGN: A novel cartilage tissue equivalent (CTE) using a no-scaffold, high-density suspension culture of neonatal porcine chondrocytes was formed on poly 2-hydroxyethyl methacrylate-treated plates for up to 16 weeks. Equilibrium aggregate modulus and hydraulic permeability were measured at 8 and 16 weeks using confined compression stress relaxation experiments. The CTE proteoglycan composition was characterized using sodium and T(1rho) magnetic resonance imaging methods after 8 weeks. RESULTS: The resultant CTE produces a biomaterial consistent with a hyaline cartilage phenotype in appearance and expression of type II collagen and aggrecan. The equilibrium aggregate modulus and permeability for the 8-week specimens were 41.6 (standard deviation (SD) 4.3) kPa and 2.85(-13) (SD 2.45(-13)) m(4)/Ns, respectively, and, for the 16-week specimens, 35.2 (SD 7.6) kPa and 2.67(-13) (SD 1.06(-13)) m(4)/Ns, respectively. Average sodium concentration of the 8-week CTE ranged from 260 to 278 mM and average T(1rho) relaxation times from 105 to 107 ms, indicating proteoglycan content similar to that of native articular cartilage. CONCLUSION: The high-density culture method produced a CTE with characteristics that approach those of native articular cartilage. The CTE mechanical properties are similar to those of the native cartilage. The CTE developed in this study represents a promising methodological advancement in cartilage tissue engineering and cartilage repair.

A method for measurement of joint kinematics in vivo by registration of 3-D geometric models with cine phase contrast magnetic resonance imaging data.

A new method is presented for measuring joint kinematics by optimally matching modeled trajectories of geometric surface models of bones with cine phase contrast (cine-PC) magnetic resonance imaging data. The incorporation of the geometric bone models (GBMs) allows computation of kinematics based on coordinate systems placed relative to full 3-D anatomy, as well as quantification of changes in articular contact locations and relative velocities during dynamic motion. These capabilities are additional to those of cine-PC based techniques that have been used previously to measure joint kinematics during activity. Cine-PC magnitude and velocity data are collected on a fixed image plane prescribed through a repetitively moved skeletal joint. The intersection of each GBM with a simulated image plane is calculated as the model moves along a computed trajectory, and cine-PC velocity data are sampled from the regions of the velocity images within the area of this intersection. From the sampled velocity data, the instantaneous linear and angular velocities of a coordinate system fixed to the GBM are estimated, and integration of the linear and angular velocities is used to predict updated trajectories. A moving validation phantom that produces motions and velocity data similar to those observed in an experiment on human knee kinematics was designed. This phantom was used to assess cine-PC rigid body tracking performance by comparing the kinematics of the phantom measured by this method to similar measurements made using a magnetic tracking system. Average differences between the two methods were measured as 2.82 mm rms for anterior/posterior tibial position, and 2.63 deg rms for axial rotation. An intertrial repeatability study of human knee kinematics using the new method produced rms differences in anterior/posterior tibial position and axial rotation of 1.44 mm and 2.35 deg. The performance of the method is concluded to be sufficient for the effective study of kinematic changes caused to knees by soft tissue injuries.