In-Vitro and In-Silico Investigation of Dynamic Compression on Cartilage Endplate Cells in Agarose

INTRODUCTION: The cartilage endplate (CEP) covers the top and bottom of the intervertebral disc (IVD) and acts to transmit compressive loads and transport water, nutrients, and waste in and out of the disc. Early cartilage endplate (CEP) degeneration is likely to play a key role in IVD degeneration, but little is known about CEP mechanobiology and its changes in degeneration. Investigating these changes is essential to elucidate how the CEP contributes to IVD pathology. METHODS: In-vitro: Bovine-tail CEP cells were expanded until passage three. Afterwards, a 1:1 mixture of CEP cells and agarose was pipetted into silicon molds to create 2% agarose and 1x107 cells/ml carriers, 6 mm diameter and 3 mm thickness, and cultured two days for phenotype recovery. Cell-agarose carriers were placed in custom-made chambers, stimulated with 10 ng/ml TGF-β1 throughout the entirety of the experiment and dynamically compressed up to 7% strain for one hour at 1.5 Hz every day for up to 14 days. Those not dynamically loaded experienced the constant weight of the chamber lid exerting ~5.1 Pa per carrier. Carriers were collected on Days 0, 7, and 14 for downstream analysis of cell viability, gene expression, and glycosaminoglycan (GAG) content. In-silico: A 2D axisymmetric porohyperelastic, compressible, Neo-Hookean finite element model (FEM) of a cell-agarose carrier was developed in Abaqus using literature-derived material properties and loaded with dynamic compression as in the in-vitro experiment. A previously developed mechanotransduction network model was used to predict protein activation levels by initial mechanoreceptor perturbations standing for dynamic compression (α5β1, αvβ3), physioosmotic pressure (TRPV4), tensile strain (αvβ5), plus chondrogenic media (TGF-β). Predicted protein activation was normalized by baseline conditions. RESULTS: After seven and 14 days of culture, cell-agarose carriers in all conditions demonstrated significantly increased expression of anabolic genes aggrecan (ACAN) 2-200x, collagen II (COL II) 31000x, and GAG/DNA content 2.5-5x, alongside decreased expression of catabolic gene matrix metalloproteinase 3 (MMP3). Reaction forces from FEM (0.07N) matched force data collected during loading (0.06N). The FEM showed that hydrostatic pressure varies from center to edge of carrier. General trends of increased/decreased gene expression and protein activation matched between experimental and network model results. DISCUSSION & CONCLUSIONS: A novel framework coupling 3D cell culture with in-silico methods is presented, in which the FEM provides details about loads experienced at each point within the carrier, while the network model uses these mechanical cues and environmental perturbations to predict protein expression and identify key proteins for future analysis. Keywords: Intervertebral disc / spine and their disorders, Biomechanics / biophysical stimuli and mechanotransductio

Direction Dependent Mass Transport Through the Cartilage Endplate

INTRODUCTION: Intervertebral Disc (IVD) Degeneration can result from chemical changes in the Cartilage Endplate (CEP) and it might suggest that pain is related to CEP weaknesses and imperfections. The CEP has a crucial role in keeping the IVD healthy by acting as the main gateway of nutrients and waste in and out of that avascular region. Yet, among the spinal tissues, CEPs receive the least amount of attention in scientific literature. The purpose of this study is to follow a combined in vitro and imaging-driven in silico approach to obtain an improved understanding of the CEP functionality regarding mass transport. METHODS: In vitro: 6 CEPs were harvested from a fresh bovine tail. Eight mm diameter biopsy samples were prepared and enabled to free swell prior to testing in Dulbecco's Modified Eagle Medium (DMEM). Each sample was fitted tightly into a silicone tube with constant inlet and outlet pressures. DMEM was passed for 10min twice in the forward and reverse direction corresponding to the flow directions into and out of the IVD in vivo respectively. The amount of passed fluid was collected to determine the flow rate. In silico: The samples were incubated in 40% Hexabrix, a contrast agent, for 48 hours and then imaged by a GE Nanotom® M nanoCT device. From the reconstructed nanoCT images, 1 mm diameter diameter subsamples were selected to create 3D models of the pore structure at different locations in the CEPs. To date, one model was meshed and imported into OpenFOAM® to perform Computational Fluid Dynamics (CFD) Simulations. The other models are still under development. RESULTS: The in vitro experiment showed that the average flow rate through that CEP samples was 6.86 mm3/sec and 4.84 mm3/sec in the forward and reverse directions respectively meaning that the reverse flow was 70.55% of the forward flow. The CFD analysis on one subsample showed that the flow rates were 15.1 mm3/sec and 10.7 mm3/sec meaning that the reverse flow was 70.86% of the forward flow. DISCUSSION & CONCLUSIONS: The results from both the in vitro experiment and the in silico simulation showed that the CEP has a tendency to resist flow differently according the direction of the flow where flow into the IVD was higher than that out of the IVD. This combination of imagebased in silico modelling with in vitro experiments is a first step towards a better quantification of the mass transport across the CEP in and out of IVDs

In-vitro investigation of compression and catabolic cytokines on human cartilage endplate cells in agarose

Introduction Intervertebral disc (IVD) degeneration is the main cause of low back cases in young adults [1]. However, the initiating and risk factors are poorly understood as it is a highly multifactorial disease. The cartilage endplate (CEP) covers the top and bottom of the IVD and acts to transmit compressive loads and transport water, nutrients, and waste in and out of the disc. [2] Early CEP degeneration is likely to play a key role in IVD degeneration, but little is known about CEP mechanobiology and its changes in degeneration. [3,4]. Investigating these changes is essential to elucidate how the CEP contributes to IVD pathology. It was hypothesized that CEP cells would behave similarly to articular chondrocytes. Thus, it was predicted that dynamic compression would be sufficient to induce anabolism, while stimulation with pro-inflammatory cytokines would induce catabolism. Material and Methods Human CEP cells were expanded until passage 3 or 4, then seeded at a density of 7.5x10 6 cells/ml into 2% agarose carriers (dimensions: 6 mm ⌀ and 3 mm height) and cultured for 5 days for phenotype recovery. Cellagarose carriers were placed in custom-made chambers, stimulated with 10 ng/ml TNF throughout the entirety of the experiment and dynamically compressed under ~7% strain for one hour at 1.5 Hz daily for up to 14 days. Carriers were collected on Days 0, 7, and 14 for downstream analysis of cell viability, metabolism, gene expression, and glycosaminoglycan (GAG) content