Recapitulating tissue elasticity can direct mesenchymal stromal cell (MSC) differentiation; however, it is unclear how substrate elasticity affects MSC metabolism. It is hypothesized MSCs subjected to stiffnesses, atypical of standard tissue culture plastic, display altered metabolic phenotypes during differentiation. In this study, such alterations in MSC metabolic profiles, based on the fluorescence lifetime of NAD(P)H, a critical co-factor in energy production, were monitored using Fluorescence lifetime imaging microscopy (FLIM) as an evaluation tool. Polyacrylamide substrates with varying stiffnesses were fabricated to model the native elasticity of cartilage and bone. MSCs cultured on these substrates exhibited potent alterations in their metabolic status over a 14-day period that were detectable as early as day 3 using FLIM. Overall, soft substrates induced a more glycolytic response after 10 days of culture that persisted at day 14 (as measured by protein-bound NAD(P)H contributions to the lifetime decay). Similarly, by day 10; MSCs on intermediate-stiffness substrates favoured glycolysis. MSCs on stiffer substrates initially displayed a glycolytic phenotype followed by a transition to oxidative phosphorylation by day 10. Staining for mineralised nodules and glycosaminoglycans verified MSCs on stiffer substrates differentiating towards an osteogenic lineage, while MSCs on intermediate substrates showed similarities with differentiated chondrocytes. Overall, it can be concluded that matrix stiffness can induce metabolic perturbations in MSCs for up to 14 days. From this research, ideal culture conditions in which the metabolics of MSCs could be manipulated to promote maximum potency could potentially be defined in the future.

The expression of the mechanosensor, integrin αvβ3, is reduced in osteoporotic bone cells compared to controls. MLO-Y4 osteocytes experience altered mechanotransduction under estrogen deficiency and it is unknown whether this is associated with defective αvβ3 expression or signalling. The objectives of this study are to (1) investigate αvβ3 expression and spatial organisation in osteocytes during estrogen deficiency, and (2) establish whether altered responses of osteocytes under estrogen deficiency correlate to defective αvβ3 expression and functionality. MLO-Y4 cells were cultured as follows: Ctrl (no added estradiol), E+ (10nM 17β-estradiol for 5 days), and Ew (10nM 17β-estradiol for 3 days and withdrawal for 2 days). Cells were cultured with/without 0.5µM IntegriSense750 (αvβ3 antagonist). Laminar oscillatory fluid flow of 1Pa at 0.5Hz was applied for 1hr. αvβ3 content was quantified using an ELISA. The location and quantity of αvβ3 and focal-adhesions was determined by immunocytochemistry. Estrogen withdrawal under static conditions led to lower cell and focal-adhesion area (p<0.05), compared to E+ cells. Fluid flow led to higher αvβ3 content (p<0.05) in all groups, compared to static counterparts, with αvβ3 blocking altering this response. Fluid flow on Ew cells had the highest αvβ3 levels (p<0.05), but αvβ3 did not localise at focal-adhesions sites. Cell morphologies were similar after treatment with the αvβ3 antagonist to the Ew group. These results suggest there are fewer functional focal-adhesion sites at which αvβ3 integrins localise to facilitate mechanotransduction. To further understand these results, we are analysing osteocyte mechanotransduction by quantifying PGE2 and gene expression (COX-2, RANKL, OPG, SOST).

Bone tissue experiences continued remodelling in response to changes in its biochemical and biophysical environment. Given the finite lifespan of osteoblasts, this continued bone formation requires replenishment from a progenitor population. Although this is largely believed to be from a skeletal stem cell population, given the limitation in in-vivo markers for this cell type, progress in demonstrating this mechanism is limited. Therefore, we characterized the LepR-Cre mouse strain and evaluated whether LepR positive cells are the progenitor population and if they contribute to the osteoblast population over time and in mechanically-induced bone formation in-vivo. Transgenic mouse strains; B6.129(Cg)-Leprtm2(cre)Rck/J to study LepR-expressing cells and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J as a reporter strain were obtained from Jackson Laboratories. Characterization studies were performed on LepR:tdTomato mice at embryonic stage (19.5dpc), 8 and 12 weeks old. Mice (12 weeks old) were subjected to compressive tibia loading with a 11N peak load for 40 cycles, every other day for 2 weeks. Histological analysis reveal that LepR is expressed from the embryonic stage in various organs including bones. LepR positive cells are found around blood vessels and on bone surfaces. Flow cytometry analysis show the amount of LepR positive cells negative for CD45 and Ter-119 markers inside the bone marrow increases over time and following tibial loading. Mechanical loading induces an increase in bone mass and bone parameters. This model allows us to track and evaluate the role of LepR positive cells as bone forming cells, and to decipher the role of these cells in mechanically-induced bone formation.

Osteoporosis affects millions globally and current anti-catabolic treatments are limited by significant side-effects. Osteoporosis arises when skeletal stem cells (SSC) no longer sufficiently replenish osteoblasts, leading to net bone loss. A key regulator of SSC behaviour is physical loading, yet the mechanisms by which SSCs sense and respond to changes in their mechanical environment are virtually unknown. Primary cilia are nearly ubiquitous ‘antennae-like’ cellular organelles that have very recently emerged as extracellular chemo/mechano-sensors and thus, are strong candidates to play an important role in regulating SSC responses in bone. This paper will demonstrate that the SSC primary cilium plays an important role in loading-induced bone formation via initial chemosensation and transduction of the potent chemokine TGFβ1 regulating SSC recruitment to the bone surface and secondly it will be shown that the primary cilium is a cAMP responsive mechanosensor directly regulating SSC mechanotransduction via localisation of adenylyl cyclase 6 to the ciliary microdomain. Finally, it will be shown that targeting the cilium therapeutically can be an effective approach to enhance both biochemical and biophysically induced SSC osteogenesis contributing to bone formation, demonstrating a novel anabolic therapy for bone loss diseases such as osteoporosis.