Purpose: Approximately 15% of the SNPs identified in the UK Biobank estimated Bone Mineral Density Genome-Wide Association Studies, were in genes preferentially expressed in osteocytes which sense mechanical loading on bone. Abnormal joint mechanics is a major risk factor for developing musculoskeletal diseases such as osteoporosis and osteoarthritis. The aim of the current study was to develop a human, 3D cell-based physiological, in vitro model of bone that can be mechanically loaded to identify mechanosensory genes. Characterising the osteocyte ‘mechanosome’ and linking to human genetic data provides an opportunity to identify risk factors for musculoskeletal diseases as well as provide new drug targets. Methods: Human MSC cells (Y201) were embedded in type I collagen gels (2mg/ml; Sigma or 4mg/ml; Advanced Biomatrix) in deformable, silicone plates to allow them to be loaded on our custom-built loading device. Cells were grown for 7-days in osteogenic conditions (DMEM, 10% FBS, 50μg/ml ascorbate-2-phosphate, 5mM β-glycerophosphate, 10nM dexamethasone). Cell viability was confirmed by live/dead assay, and osteocyte phenotype by RTqPCR and immunostaining for E11, sclerostin (SOST) and DMP-1. On day-7, embedded cells in silicone plates were subjected to a single physiological (500 μstrain) or pathophysiological (5000 μstrain) load (10Hz, 3000 cycles; TE Instruments). Media was collected 1- and 24 hours post-load and analysed for indicators of bone turnover (osteoprotegerin (OPG), ELISA, Abcam) and inflammation (Merck Milliplex multiplex panel). Data was analysed by GLM ANOVA and Fisher’s post-hoc test (Minitab, n=3/treatment). Results: Y201 MSC cells became dendritic within one hour of embedding in 3D collagen gels and remained viable and osteocyte-like for 7-days in both 2mg/ml or 4mg/ml collagen in silicone loading plates. RNA extracted at 7-days was of good quality (RIN scores 6-9). RTqPCR analysis revealed cells expressed markers of mature osteocytes (E11, DMP-1, SOST) as well as expressing osteoprotegerin, osteocalcin and type I collagen. Positive immunolocalisation of sclerostin and DMP-1 protein confirmed osteocyte-like phenotype (fig 1). OPG release was reduced 1hr after physiological (1.7-fold, p=0.07) and pathophysiological (3.8-fold, p=0.005) loading (fig 2), but increased after loading 24hrs later (phys 7-fold, p=0.004; pathophys 2.6-fold, p=0.05). Both loading regimes abolished release of GM-CSF and RANTES and reduced release of MCP-1 (1.4-fold), IL-6 (1.4-fold) and IP-10 (2.7-fold) (p<0.05, fig 3). Conclusions: Y201 cells, a human MSC immortalised cell line, were successfully differentiated to osteocytes, maintaining viability and phenotype in silicone plates over 7-days in culture in different concentrations of telocollagen with variable matrix stiffness. The assessment of the osteocytes’ mechanical response with a focus on factors that contribute to disease, such as bone remodelling and inflammation provides a proof of concept that this model can provide mechanistic data to improve understanding of the role of load in disease progression. Since underlying molecular mechanisms in abnormal mechanical load are not clear, there is a need for humanised, cell-based models to examine molecular pathways activated by mechanical load to identify risk factors and new therapeutic approaches.