0409 - HiT-MACE: A High-Throughput Mechanical Activator for Cartilage Engineering to Study the Early Cartilage Response to Physiological and Supra-physiological Loads
Year: 2019
Type: other
Abstract: INTRODUCTION: Articular cartilage is crucially influenced by mechanical loading in development, health, and disease. Regenerative medicine approaches need to incorporate mechanical loading as a key component to drive tissue repair as well as to generate engineered constructs that can substitute the damaged cartilage [1]. Recently, regenerative rehabilitation has been proposed as the use of regenerative medicine knowledge to inform physical therapy to support and enhance repair after surgery [1]. However, notwithstanding a large body of knowledge on cartilage mechanobiology, our knowledge on the ideal mechanical conditions that promote cartilage repair and on the specific effects of loading in health and disease are still limited. To address this issue, we have developed a novel device for the high throughput compressive loading of native and engineered cartilage called HiT-MACE (High Throughput Mechanical Activator for Cartilage Engineering) to allow for the screening of a high number of samples under different conditions (Fig.1). With HiT-MACE we are able to apply cyclic loads in the physiologic range (e.g., equivalent to walking and normal daily activity) and supra-physiologic range (e.g., injurious impacts or long marches with a heavy backpack as in basic military training) to up to 24 samples in one single run. In this work, we validated the system first on model samples, then we studied the short term, anabolic response of cartilage to physiological loading, as well as to supra-physiological loading.METHODS: Device: The HiT-MACE device is composed of pistons attached to six compact actuators (L2018S0604-T3,5X1-25, Nanotech) equipped with force sensors (FlexiForce, Tekscan) to provide force feedback during loading (Fig.1). The base hosting the 24 well plate with the samples is operated by two x,y motors (Velmex), so that all samples are mechanically activated by the pistons in sequence, six at a time. A custom lid equipped with a flexible membrane as well as pistons and piston guides were designed in Solidworks and manufactured by 3D printing with a Perfactory Vida printer and proprietary, biocompatible e-shell 450 resin (EnvisionTek) (Fig.1). A National Instrument DAQ board connects the system to a computer and a Labview user interface.Device Validation: We compared the Youngu2019s modulus of a PDMS (n=24) and 4% (w/v) agarose (n=24) cylinders, synthetic materials with mechanical properties in the range of cartilage, measured with HiT-MACE and with the Bose (now TAinstrument) ElectroForce 3230 mechanical tester.Cartilage early response to loads: Articular cartilage cylinders of 4mm in diameter of articular cartilage were harvested from freshly euthanized pigs from a local abattoir. Four to eight donor animals were used for each experiment. After 48h equilibration in serum-free, TGF-u03b2-free medium (DMEM, 2% penicillin/streptomycin, ITS, and ascorbate), samples were subject to physiologic cyclic loading (12% strain at 1Hz) for 30 minutes. A second set of samples was subject instead to supra-physiological loading (45% cyclic strain) for 30 minutes. All samples were then analyzed for gene expression by qRT-PCR of the extracted RNA (porcine genes: ACAN, BMP2, COL2, ALK5, PAI1, SMAD3, TGF-u03b21, COLX, ID1, MMP13) and by immunohistochemistry and histology. The supra-physiological loading results were compared to the response to IL-1u03b2 exposure as an inflammation-based model of tissue damage. Statistical analysis: Results were compared by either t-Test or ANOVA following by a post hoc Tukey test with significance set at p=0.05.RESULTS: No significant difference was found between Youngu2019s moduli measured by the HiT-MACE and the ElectroForce mechanical tester, confirming the effectiveness of HiT-MACE. However, testing of 24 samples with HiT-MACE took 30 minutes and was automated, while with ElectroForce each sample had to be manually tested for a total of 5 hours work. For cartilage analysis, each run of the HiT-MACE comprised tissues from 4 porcine donors allowing for a rapid samples turnaround. Physiological loading induced upregulation of ACAN, COL2, BMP2, ALK5, SMAD3, TGF-u03b21, and ID-1 (8 donors in 2 HiT-MACE runs, p=0.05 or less) (Fig. 2), while supraphysiological loading upregulated only BMP2, ALK5, and TGF-u03b21, and IL-1u03b2 exposure induced upregulation of BMP2, ALK5, and MMP13. Immunohistochemistry confirmed these gene expression in the cells within cartilage sections. DISCUSSION: The use of a high throughput system such as our HiT-MACE allowed to rapidly generate a sizeable amount of data from multiple donors and to reach significance in a very short time. Cartilage subject to physiological loading rapidly responds with increased gene expression of anabolic markers along the TGF-u03b21 pathway (e.g., COL2, ACAN, SMAD3, etc.) (Fig. 2), confirming what previously reported by Madej et al. [2] Notably, no TGF-u03b21 or serum were present in the media, so the changes in gene expression are primarily mechanobiologically driven. Supraphysiological loading, in the short term, does not have a marked anabolic or catabolic response, whereas IL-1u03b2 clearly initiates a catabolic response as expected. Long term effects of both will be the subject of further studies, as well as the effects of sequential supra-physiological followed by physiological loading (modelling a rehabilitation exercise/light pace walk after excessive weight bearing).SIGNIFICANCE/CLINICAL RELEVANCE: High throughput mechanical loading of cartilage and engineered constructs allows to generate large volumes of data and samples to effectively explore cartilage response to physiological and supraphysiological loading and to engineer more effective repair and rehabilitation strategies.REFERENCES: [1] Gottardi R. and Stoddart MJ, J Am Acad Orthop Surg (2018) 26(15):e321-e323. [2] Madej W, et al. Osteoarthr. Cartil. (2016) 24(10):1807-1815. ACKNOWLEDGEMENTS: Ri.MED Foundation, AR3T through NIH grant no. P2CHD086843, NIH (UG3TR002136)
Authors: Riccardo Gottardi, Vincenzo La, Rocky Tuan
Language: English
Subfield: Rheumatology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W4391553274