A Novel High-Throughput System for the Mechanical Analysis of Engineered Cartilage Constructs 1,2Cosgrove, BD; 2Lewis, G; 2Winarto, B; 2Wong, J; 1Dodge, GR; +1,2Mauck, RL

1Department of Orthopaedic Surgery, 2Department of Bioengineering, University of Pennsylvania, Philadelphia, PA lemauck@mail.med.upenn.edu

INTRODUCTION While the field of cartilage tissue engineering has made marked progress over the last decade (achieving near native levels for chondrocyte based engineered cartilage [1, 2]) one major limitation is the lengthy time required to mechanically evaluate engineered constructs. Additionally, uniform mechanical impaction of these materials has not been possible on a large scale. Given the high cost of mechanical testing systems, throughput is a significant limitation. Most mechanical testing systems employ a single actuator and load cell, necessitating sample-by-sample processing. This is particularly evident when considering the large number of variables and replicates needed in any given study. For example, in one recent study [3], we performed 60 independent mechanical tests on a biweekly basis, equating to ~100 hours of labor at each time point. Given the large number of variables to be considered, low throughput sample-by-sample mechanical testing limits experimental design and hampers progress. High throughput screening (HTS) methods developed in the pharmaceutical industry provide for rapid evaluation of cell responses to stimuli, enabling the screening of thousands of factors simultaneously in an economical and reproducible fashion. We have developed robotic HTS methods to evaluate biochemical content during mesenchymal stem cell differentiation (with screening of small molecule libraries) [4]. Here, we extend this work to develop a system for high throughput mechanical screening (HTMS) of engineered cartilage constructs. The device can likewise be used to evaluate outcomes after controlled mechanical perturbation (normal and supra-physiologic) of cartilage-like constructs.

METHODS Device Components and Design: A custom HTMS device was designed using an IFSR resistive multi-touch sensor [Touchco Inc., New York, NY] mounted in an apparatus that provides linear movement in the vertical axis (Fig 1). Samples were placed in a 24-well tissue culture plate that was then inserted into the apparatus. The IFSR sensor measures reaction forces generated from sample compression via 24 cylindrical PTFE indenter platens (Ø 9.525 mm, h=59.71 mm ± 0.06 mm). A traditional mechanical testing system (Instron 5543) provided overall compressive displacement to the high throughput adapter (Fig 1). Test Protocol Development: Samples were evaluated by establishing a small pre-load to ensure that all indenters were in contact with the IFSR sensor. Subsequently, a nominal 10% compressive strain was applied. With loading, the sensor sampled data at a frequency of 1Hz. Equilibrium force readings were base-lined against the initial force readings; this protocol mitigates the influence of slight differences in sample or indenter heights. Prior to testing, the IFSR sensor was calibrated to ensure accurate conversion of the relative force units outputted to engineering units (Newtons). Collection of the force data from the IFSR sensor was controlled using a custom C# program and the Touchco API. During data collection, each well of the test plate was assigned a corresponding 4 x 4 sensile area (~256 mm2 sensor area/well for a 24 well plate), and total force across each well was tabulated (Fig 2). All data visualization was performed using MATLAB [Mathworks Inc., Natick, MA]. System Validation and Sample Testing: Multiple PDMS cylinders (Ø 8mm, h=6.37mm) were tested simultaneously in the HTMS device followed by individual analysis using an Instron (with the same testing parameters). To illustrate the capacity of the device to capture time-dependent properties, bovine cartilage cylinders (Ø 4mm, h=3.17mm) were evaluated by sequential stress-relaxation (5% strain, 20 min hold, 10% strain, 40 min hold).

RESULTS The HTMS testing device accurately captured both equilibrium and dynamic reaction forces from compression of an array of synthetic and natural materials (Fig 2). The conversion factor for each test was determined through direct compression of the sensor through the platens. In validation studies, the modulus of PDMS samples tested individually (~3.8 MPa, n=8, Fig 3A) was slightly higher than that found for samples tested simultaneously using the HTMS system (~3.4 MPa, n=8, within 10% of the individual measures). As the HTMS will be used for primary screening, slightly lower thresholds for accuracy are acceptable, as secondary screens follow on from ‘hits’ identified in these primary screens. When the device was used to mechanically compress articular cartilage, the IFSR sensor captured the time-dependent stress-relaxation response (Fig 3B).

Figure 1: HTMS system. (Left) Image of HTMS apparatus with the sensor platen removed. (Right) Schematic of HTMS apparatus in cross-section.

Figure 2: Color maps of IFSR force data resulting from a 10-sample HTMS test, displayed in both 2D (A) and 3D (B). Color scale is maintained.

Figure 3: (A) Equilibrium moduli of PDMS samples tested simultaneously on the HTMS system and individually via Instron (mean ± SD, n=8) (B) Stress-relaxation response using the HTMS for articular cartilage over a series of compressive steps.

DISCUSSION We have developed a HTMS tool for analysis of native and engineered tissues. In this prototype device, transient and equilibrium reaction forces were acquired simultaneously from an array of up to 24 samples. The device allows for parallel and reliable mechanical evaluation, and does so in a cost effective manner (the most expensive component, the sensor, costs ~$500). Others have developed systems that allow for multiple sample analysis. For example, the MATE system [5] was designed to both measure and stimulate six samples at one time. That device, while useful, is limited by cost, as it relies on a one sample to one load cell/actuator ratio. Our current HTMS device can evaluate 24 samples at the same time, but can be easily scaled to accommodate 96- and 384-well plate designs. The device is also compatible with other electronic pressure sensitive films (i.e., Tekscan). With transition to higher numbers, small molecule screening with mechanical outcomes will be possible, as will complex combinatorial assays examining growth factor dosage and combinations in parallel. Analysis via HTMS can be further combined with our existing in-well HTS assays of biochemical content [4]. Moreover, the system could be modified to operate sterilely, so as to analyze small molecule interactions with mechanical stimuli (normal and/or injurious). This validated testing platform will accelerate evaluation of molecular response and mechanical properties in cartilage tissue engineering, and may help to identify disease-modifying agents in post-traumatic OA.

SIGNIFICANCE Cartilage tissue engineering has been hampered by the low throughput and high cost of traditional mechanical testing systems. Here, we present a novel and economical system for high throughput mechanical screening (HTMS) of engineered tissues, enabling small molecule screening and combinatorial analyses. ACKNOWLEDGEMENTS This work was supported by the NIH (R01 EB008722) and the AO Foundation Acute Cartilage Injury Consortium. REFERENCES 1) Byers+ OAC 2008. 2) Lima+ OAC 2007. 3) Erickson+ TE 2009. 4) Huang+ ABME 2010. 5) Lujan+ TE 2011.

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Paper No. 0286 • ORS 2012 Annual Meeting