Lena R. Bartell

Measuring microscale strain fields in articular cartilage during rapid impact reveals thresholds for chondrocyte death and a protective role for the superficial layer

Articular cartilage is a heterogeneous soft tissue that dissipates and distributes loads in mammalian joints. Though robust, cartilage is susceptible to damage from loading at high rates or magnitudes. Such injurious loads have been implicated in degenerative changes, including chronic osteoarthritis (OA), which remains a leading cause of disability in developed nations. Despite decades of research, mechanisms of OA initiation after trauma remain poorly understood. Indeed, although bulk cartilage mechanics are measurable during impact, current techniques cannot access microscale mechanics at those rapid time scales. We aimed to address this knowledge gap by imaging the microscale mechanics and corresponding acute biological changes of cartilage in response to rapid loading. In this study, we utilized fast-camera and confocal microscopy to achieve roughly 85 µm spatial resolution of both the cartilage deformation during a rapid (~3 ms), localized impact and the chondrocyte death following impact. Our results showed that, at these high rates, strain and chondrocyte death were highly correlated (p<0.001) with a threshold of 8% microscale strain norm before any cell death occurred. Additionally, chondrocyte death had developed by two hours after impact, suggesting a time frame for clinical therapeutics. Moreover, when the superficial layer was removed, strain - and subsequently chondrocyte death - penetrated deeper into the samples (p<0.001), suggesting a protective role for the superficial layer of articular cartilage. Combined, these results provide insight regarding the detailed biomechanics that drive early chondrocyte damage after trauma and emphasize the importance of understanding cartilage and its mechanics on the microscale.

Multiscale Strain as a Predictor of Impact-Induced Fissuring in Articular Cartilage

Mechanical damage is central to both initiation and progression of osteoarthritis (OA). However, specific causal links between mechanics and cartilage damage are incompletely understood, which results in an inability to predict failure. The lack of understanding is primarily due to the difficulty in simultaneously resolving the high rates and small length scales relevant to the problem and in correlating such measurements to the resulting fissures. This study leveraged microscopy and high-speed imaging to resolve mechanics on the previously unexamined time and length scales of interest in cartilage damage, and used those mechanics to develop predictive models. The specific objectives of this study were to: first, quantify bulk and local mechanics during impact-induced fissuring; second, develop predictive models of fissuring based on bulk mechanics and local strain; and third, evaluate the accuracy of these models in predicting fissures. To achieve these three objectives, bovine tibial cartilage was impacted using a custom spring-loaded device mounted on an inverted microscope. The occurrence of fissures was modulated by varying impact energy. For the first objective, during impact, deformation was captured at 10,000 frames per second and bulk and local mechanics were analyzed. For the second objective, data from samples impacted with a 1.2 mm diameter rod were fit to logistic regression functions, creating models of fissure probability based on bulk and local mechanics. Finally, for the third objective, data from samples impacted with a 0.8 mm diameter rod were used to test the accuracy of model predictions. This study provides a direct comparison between bulk and local mechanical thresholds for the prediction of fissures in cartilage samples, and demonstrates that local mechanics provide more accurate predictions of local failure than bulk mechanics provide. Bulk mechanics were accurate predictors of fissure for the entire sample cohort, but poor predictors of fissure for individual samples. Local strain fields were highly heterogeneous and significant differences were determined between fissured and intact samples, indicating the presence of damage thresholds. In particular, first principal strain rate and maximum shear strain were the best predictors of local failure, as determined by concordance statistics. These data provide an important step in establishing causal links between local mechanics and cartilage damage; ultimately, data such as these can be used to link macro- and micro-scale mechanics and thereby predict mechanically mediated disease on a subject-specific basis.

Local and global measurements show that damage initiation in articular cartilage is inhibited by the surface layer and has significant rate dependence

Cracks in articular cartilage are a common sign of joint damage, but failure properties of cartilage are poorly understood, especially for damage initiation. Cartilage failure may be further complicated by rate-dependent and depth-dependent properties, including the compliant surface layer. Existing blunt impact methods do not resolve local cartilage inhomogeneities and traditional fracture mechanics tests induce crack blunting and may violate underlying assumptions of linear elasticity. To address this knowledge gap, we developed and applied a method to indent cartilage explants with a sharp blade and initiate damage across a range of loading rates (strain rates 0.5%/s-500%/s), while recording local sample deformation and strain energy fields using confocal elastography. To investigate the importance of cartilages compliant surface, we repeated the experiment for samples with the surface removed. Bulk data suggest a critical force at which the tissue cuts, but local strains reveals that the deformation the sample can sustain before reaching this force is significantly higher in the surface layer. Bulk and local results also showed significant rate dependence, such that samples were easier to cut at faster speeds. This result highlights the importance of rate for understanding cracks in cartilage and parallels recent studies of rate-dependent failure in hydrogels. Notably, local sample deformation fields were well fit by classical Hookean elasticity. Overall, this study illustrates how local and global measurements surrounding the initiation of damage in articular cartilage can be combined to reveal the importance of cartilages zonal structure in protecting against failure across physiologically relevant loading rates.

Microscale frictional strains determine chondrocyte fate in loaded cartilage

Mounting evidence suggests that altered lubricant levels within synovial fluid have acute biological consequences on chondrocyte homeostasis. While these responses have been connected to increased friction, the mechanisms behind this response remain unknown. Here, we combine a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, microscale shear strain, and acute, adverse cellular responses. Our incorporation of cell-scale strain measurements reveals that elevated friction generates high shear strains localized near the tissue surface, and that these elevated strains are closely associated with mitochondrial dysfunction, apoptosis, and cell death. Collectively, our data establish two pathways by which chondrocytes negatively respond to friction: an immediate necrotic response and a longer term pathway involving mitochondrial dysfunction and apoptosis. Specifically, in the surface region, where shear strains can exceed 0.07, cells are predisposed to acute death; however, below this surface region, cells exhibit a pathway consistent with apoptosis in a manner predicted by local shear strains. These data reveal a mechanism through which cellular damage in cartilage arises from compromised lubrication and show that in addition to boundary lubricants, there are opportunities upstream of apoptosis to preserve chondrocyte health in arthritis therapy.

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling.