Abstract
Introduction and Objective
Bone remodelling is a continuous process whereby osteocytes regulate the activity of osteoblasts and osteoclasts to repair loading-induced microdamage. While many in vitro studies have established the role of paracrine factors (e.g., RANKL/OPG) and cellular pathways involved in bone homeostasis, these techniques are generally limited to two-dimensional cell culture, which neglects the role of the native extracellular matrix in maintaining the phenotype of osteocyte. Recently, ex vivo models have been used to understand cell physiology and mechanobiology in the presence of the native matrix. Such approaches could be applicable to study the mechanisms of bone repair, whilst also enabling exploration of biomechanical cues. However, to date an ex vivo model of bone remodelling in cortical bone has not been developed. In this study, the objective was to develop an ex vivo model where cortical bone was subjected to cyclic strains to study the remodelling of bone.
Materials and Methods
Ex vivo model of bone remodelling induced by cyclic loading: At the day of culling, beam-shape bovine bone samples were cut and preserved in PBS + 5% Pen/Strep + 2 mM L-Glut overnight at 37°C. Cyclic strains were applied with a three-point bend system to induce damage with a regime at 16.66 mm/min for 5,000 cycles in sterile PBS in Evolve® bags (maximum strain 6%). A control group was cultured under static conditions.
Metabolic activity: Alamar Blue assays were performed after 1 and 7 days of ex vivo culture for each group (Static, Loaded) and normalized to weight.
Bone remodelling: ALP activity was assessed in the media at day 1 and 7. After 24 hours cell culture conditioned media (CM) was collected from each group and stored at −80°C. RAW264.7 cells were cultured with CM for 6 days, after which the samples were stained for TRAP, to determine osteoclastogenesis, and imaged.
Histomorphometry: Samples were cultured with calcein for 3 days to label bone formation between day 4 and 7. Fluorescent images were captured at day 7. μCT scanning was performed at 3 μm resolution after labelling samples with BaSO4 precipitate to quantify bone damage.
Results
Bone was sectioned and cultured to maintain live osteoblasts and osteocytes. CM that was obtained 24 hours after cyclic loading and added to RAW264.7 cells cultures, resulted in significantly increased osteoclastogenic potential compared to that from static samples (4.245±1.65% vs 0.88±0.48%, p<0.001). Calcein and HE staining indicated the presence of structures similar to bone remodelling cones in both groups after 7 days of culture. Also, 7 days post-loading, matrix microdamage in the stimulated area, detected with the BaSO4 precipitate, were not significantly increased under the load point in loaded samples (0.11±0.05% of bone volume), while at the support areas it was significantly higher (0.2387±0.06%, p<0.001) compared to the static (0.062±0.02%).
Conclusions
This study demonstrates that (1) cyclic strains applied on ex vivo bovine cortical bone successfully induced remodelling as characterized by the formation of bone resorption cones, along with an increase of osteoclast formation, and (2) there was an induction of microdamage post loading as shown by the significant increases in microdamage labelled. This supports previous in vivo studies with an increase in osteoclastogenesis up to 7 days post loading. This is the first evidence of the development of an ex vivo model to study osteon remodelling that could be applied to study bone physiology and repair.
