We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability-induced osteoclast differentiation and peri-implant bone loss. MLO-Y4-osteocytes were mechanically stimulated for 1h by fluid shear stress using regimes simulating: (i) supraphysiological loading in the peri-prosthetic interface (2.9 +/- 2.9Pa, 1Hz, square wave); (ii) physiologic loading in the cortical bone (0.7 +/- 0.7Pa, 5Hz, sinusoidal wave); and (iii) stress shielding. Cellular morphological parameters, membrane-bound RANKL expression, gene expression influencing osteoclast differentiation, nitric oxide release and caspase 3/7-activity were determined. Either Mouse bone marrow cells were cultured on top of loaded osteocytes or osteocyte-conditioned medium was added to bone marrow cells. Osteoclast differentiation was assessed after 6 days. We found that osteocytes subjected to supraphysiological loading showed similar morphology and caspase 3/7-activity compared to simulated physiological loading or stress shielding. Supraphysiological stimulation of osteocytes enhanced osteoclast differentiation by 1.9-fold compared to physiological loading when cell-to-cell contact was permitted. In addition, it enhanced the number of osteoclasts using conditioned medium by 1.7-fold, membrane-bound RANKL by 3.3-fold, and nitric oxide production by 3.2-fold. The stimulatory effect of supraphysiological loading on membrane-bound RANKL and nitric oxide production was higher than that achieved by stress shielding. In conclusion, the in vitro model developed recapitulated the catabolic biological situation in the peri-prosthetic interface during instability that is associated with osteoclast differentiation and enhanced RANKL expression. The model thus provides a platform for pre-clinical testing of pharmacological interventions with potential to stop instability-induced bone implant loosening. (c) 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1425-1434, 2018.

Mechanical instability of bone implants stimulate osteoclast differentiation and peri-implant bone loss, leading to prosthetic loosening. It is unclear which cells at the periprosthetic interface transduce mechanical signals into a biochemical response, and subsequently facilitate bone loss. We hypothesized that mechanical overloading of hematopoietic bone marrow progenitor cells, which are located near to the inserted bone implants, stimulates the release of osteoclast-inducing soluble factors. Using a novel in vitro model to apply mechanical overloading, we found that hematopoietic progenitor cells released adenosine triphosphate (ATP) after only 2min of mechanical loading. The released ATP interacts with its specific receptor P2X7 to stimulate the release of unknown soluble factors that inhibit (physiological loading) or promote (supraphysiological loading) the differentiation of multinucleated osteoclasts derived from bone marrow cultures. Inhibition of ATP-receptor P2X7 by Brilliant Blue G completely abolished the overloading-induced stimulation of osteoclast formation. Likewise, stimulation of P2X7 receptor on hematopoietic cells by BzATP enhanced the release of osteoclastogenesis-stimulating signaling molecules to a similar extent as supraphysiological loading. Supraphysiological loading affected neither gene expression of inflammatory markers involved in aseptic implant loosening (e.g., interleukin-1 (IL-1), IL-6, tumor necrosis factor-, and PTGES2) nor expression of the osteoclast modulators receptor activator of nuclear factor -B ligandand osteoprotegerin. Our findings suggest that murine hematopoietic progenitor cells are a potential key player in local mechanical loading-induced bone implant loosening via the ATP/P2X7-axis. Our approach identifies potential therapeutic targets to prevent prosthetic loosening.