INTRODUCTION

The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. Glutamate signalling has been implicated in osteoarthritic bone changes and AMPA/kainate glutamate receptor (GluR) antagonists alleviate degeneration in rodent models of osteoarthritis. We investigated whether glutamate signalling molecules are mechanically regulated in a human, cell-based 3D model of bone.

METHODS

Human Y201 MSC cells embedded in 3D type I collagen gels (0.05 × 106 cell/gel) differentiated to osteocytes were mechanically loaded in silicone plates (5000 µstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1-hr post load was quantified by RTqPCR and RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). Differentially expressed GluRs and glutamate transporters (GluTs) were identified using DEseq2 analysis on normalised count data. Genes were considered differentially expressed if >2 fold change and FDR p<0.05.

RESULTS

Cells expressed mature osteocyte markers (E11, sclerostin, DMP-1). DEseq2 analysis, revealed 981 mechanically regulated genes. Mechanical loading upregulated kainate GluRs, GRIK2 (1.6 fold, p=0.024) and GRIK5 (4.2 fold, p=0.045); the NMDA GluR GRIN3B (3.25 fold, p=0.047) and the GluT SLC1A1 (3 fold, p=0.037). Conversely, AMPA GRIA3, NMDA GluRs GRIN2A&C, and the GluT SLC1A2 were down regulated by 50–60%, although not significant. Kainate GRIK3&4; AMPA GRIA2, NMDA GRIN1, and GluTs SLC1A6&A7 were not expressed in control or loaded osteocytes, whereas GluRs (GRIK1, GRIA1&4, GRIN2B&2D&3A) and GluT SLC1A3 were expressed but not regulated by mechanical loading.

DISCUSSION

Mechanical loading of human osteocytes in 3D revealed that they regulated expression of glutamate receptors and transporters. This is consistent with our observation that mechanical perturbation after joint injury in rodent models of OA regulates glutamatergic signalling in the bone thus linking mechanical stimuli to inflammatory and nociceptive pathways mediated by glutamate receptors.

Declaration of Interest

(a) fully declare any financial or other potential conflict of interest