Time Scales in Cartilage Impact Injury and Chondrocyte Death

Transcription

1 Time Scales in Cartilage Impact Injury and Chondrocyte Death Lena R. Bartell, Michelle L. Delco, DVM, Lawrence J. Bonassar, Lisa A. Fortier, Itai Cohen. Cornell University, Ithaca, NY, USA. Disclosures: L.R. Bartell: None. M.L. Delco: None. L.J. Bonassar: None. L.A. Fortier: None. I. Cohen: None. Introduction: Osteoarthritis (OA) resulting from acute injury has been heavily studied due to its prevalence and predictable longterm development. A large range of times scales are relevant to the disease development a mechanical catalyst may only last a few milliseconds, while symptoms take decades to emerge. Current in vitro OA models typically observe the effects of injury on the scale of days or longer [1,2]. Additionally, existing research on injury mechanics has focused on slow or equilibrated loading at video frame rates, which cannot track cartilage effectively during true pathological loading [3,4]. As a result, little is known about the mechanical environment inside articular cartilage during injury or its earlytime response. The purpose of this study was to design and implement techniques to observe, ex vivo, the local mechanics of articular cartilage during rapid mechanical impact and the resulting earlytime cell response. Such techniques allow indepth exploration of PTOA initiation and provide a direct link between the mechanics of trauma and the earliest biomechanical changes in the tissue. In particular, our method allows for the local (~100 µm) tissue deformation during rapid impact to be correlated with the local, timedependent decrease in cell viability following that impact. Methods: Sample Preparation: Cylindrical cartilage plugs (6 mm diameter, 23 mm deep) were harvested from the tibial plateau of 13 day old bovine. Samples were immediately transferred to phenolredfree Dulbecco's Modified Eagle Medium with antibiotics and 10% FBS. Imaging rapid deformation: We designed a springloaded impact device to integrate with an inverted confocal microscope and a highspeed video camera recording at 1,000 frames per second (v7.1, Vision Research, Wayne, NJ). 10 plugs were cut into two hemicylinders; one half was impacted, the other was a nonimpacted control. All samples were stained with DTAF, an allprotein stain. Using a wire mesh template (TWP Inc, Berkeley, CA), a 120 μm grid was photobleached. The deep surface of each sample was then glued to a stable back plate so that the depthprofile could be viewed from below. The impact tip, a 0.8 mm diameter rod, was mounted vertically but driven horizontally toward the impact samples such that imaging from below showed the rod s circular cross section approaching the samples. Impacts had a peak force of about 12 N and lasted 8 ms. During impact, local deformation of the photobleached grid was observed by the fast camera and tracked using custom MATLAB code, producing strain fields with a spatial resolution of roughly 85 μm.

2 Cell death after impact: During impact testing, each sample was suspended in a bath of 2 μm ethidium homodimer (EthD) to stain dead cells. Confocal imaging was used to track the locations of these cells before and for three hours following impact, in tenminute intervals. The control sample was then frozen for at least 24hours, restained, and imaged again to get a total cell count. This was used to compute the local evolution of cell viability following injury. Correlating mechanics and cell death: Local impact deformation was correlated with the local decrease in viability following impact. The known pixel size and impact location were used to transform between the fast camera and confocal images. Fast camera grid cross locations in the undeformed configuration (red points in Fig. 1A, 120 µm nominal spacing) were thus projected onto the confocal image of the impacted sample at 3 hours postimpact. These grid points were then used as bin boundaries for calculating the local viability. This was then used to calculate the probability of cells dying within 3 hours of impact. This probability was correlated with the norm and the shear magnitude of the local Lagrange strain field at the time of peak deformation. Results: Cartilage deformation was tracked during rapid impact (Fig. 1AB). Local strain fields (Fig. 1CD) show that deformation is concentrated in the first μm from the articular surface and extends more laterally than with depth. Fig. 2 shows the local probability of a cell dying as a function of depth from the surface and time after impact. Points above 200 µm were discarded as invalid because handling induced nearly 100% cell death in the very superficial layer of all samples. Cells within 700 µm of the surface are affected most prominently, while the time evolution implies a timescale of roughly 12 hours after impact for the earlytime cell response to occur. Correlations between local strain and local probability of death are shown in Fig. 3. Discussion: As revealed in local strain maps, the deformation of cartilage during impact is largely confined near the surface. Recent experiments showed that the surface of cartilage is much more compliant and absorbs more energy than the bulk [5], which is in agreement with these strain patterns. To our knowledge, this is the first observation of local cartilage motion during a pathological impact and is a fundamental shift in the approach to understanding cartilage injury. In particular, this method opens the door to correlate local mechanics during injury to a tissue s early time biological response. The spatial and temporal evolution cell viability after impact shows a peak probability of death near the surface, but the majority effect extends about 500 µm into the tissue. This roughly corresponds to the location of the transition zone in cartilage, where the tibial plateau tissue is most compliant [5,6]. Moreover, this peak is not insignificant it is more than a 50% effect over control. Correlation with Lagrange strain norm is highest and the nonzero xaxis intercept implies a strain threshold for cell death around 5% (at this rate). Overall, this technique for rapid impact microscopy is a novel approach to understanding cartilage impact in detail and presents the firstlook at local deformation during cartilage injury as well as the earlytime spatial and temporal evolution of cell death. Significance:

3 Injury of articular cartilage and PTOA initiation are important and active research topics relevant to both the basic and clinical sciences. This work presents a novel view of cartilage during and immediately following a pathological injury, highlighting the injury mechanics and earlytime biological response, as well as the potential implications for therapy.

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5 ORS 2015 Annual Meeting Paper No: 0251