Thermally Induced Osteocyte Damage Initiates a Remodelling Signaling Cascade

March Thermally Induced Osteocyte Damage Initiates a Remodelling Signaling Cascade Eimear B. Dolan 0 1 2 Matthew G. Haugh 0 1 2 Muriel C. Voisin 0 1 2 David Tallon 0 1 2 Laoise M. McNamara 0 1 2 0 1 Biomechanics Research Centre (BMEC), Biomedical Engineering, College of Engineering and Informatics, National University of Ireland , Galway, Ireland , 2 National Centre for Biomedical Engineering Science (NCBES), National University of Ireland , Galway, Ireland, 3 Stryker Ireland, Carrigtwohill, Cork , Ireland 1 Funding: The work was supported by National University of Ireland , Galway Fellowship Scheme (ED LMcN) , The National University of Ireland Travelling Scholarships in Engineering (ED LMcN), and the European Research Council (ERC) (under grant no.258992;BONEMECHBIO) (LMcN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Denise Pires de Carvalho , Universidade Federal do Rio de Janeiro, BRAZIL Thermal elevations experienced by bone during orthopaedic procedures, such as cutting and drilling, exothermal reactions from bone cement, and thermal therapies such as tumor ablation, can result in thermal damage leading to death of native bone cells (osteocytes, osteoblasts, osteoclasts and mesenchymal stem cells). Osteocytes are believed to be the orchestrators of bone remodeling, which recruit nearby osteoclast and osteoblasts to control resorption and bone growth in response to mechanical stimuli and physical damage. However, whether heat-induced osteocyte damage can directly elicit bone remodelling has yet to be determined. This study establishes the link between osteocyte thermal damage and the remodeling cascade. We show that osteocytes directly exposed to thermal elevations (47C for 1 minute) become significantly apoptotic and alter the expression of osteogenic genes (Opg and Cox2). The Rankl/Opg ratio is consistently down-regulated, at days 1, 3 and 7 in MLO-Y4s heat-treated to 47C for 1 minute. Additionally, the pro-osteoblastogenic signaling marker Cox2 is significantly up-regulated in heat-treated MLO-Y4s by day 7. Furthermore, secreted factors from heat-treated MLO-Y4s administered to MSCs using a novel co-culture system are shown to activate pre-osteoblastic MSCs to increase production of the pro-osteoblastic differentiation marker, alkaline phosphatase (day 7, 14), and calcium deposition (day 21). Most interestingly, an initial pro-osteoclastogenic signaling response (increase Rankl and Rankl/Opg ratio at day 1) followed by later stage pro-osteoblastogenic signaling (down-regulation in Rankl and the Rankl/Opg ratio and an up-regulation in Opg and Cox2 by day 7) was observed in non-heat-treated MLO-Y4s in co-culture when these were exposed to the biochemicals produced by heat-treated MLO-Y4s. Taken together, these results elucidate the vital role of osteocytes in detecting and responding to thermal damage by means of thermally induced apoptosis followed by a cascade of remodelling responses. - During orthopedic intervention, bone and the surrounding soft tissue can be exposed to elevated temperatures arising from surgical cutting and drilling, exothermal reactions from bone cement during curing, and thermal therapies such as tumor ablation. Temperatures up to 100C have been reported to arise in surrounding bone tissue as a result of the cutting and drilling processes during orthopaedic surgeries [1,2,3]. Polymethylmethacrylate polymerisation can result in temperatures ranging from 40 to 100C [4,5]. Thermal ablation has been widely used treat a number of bone diseases including malignant bone tumors [6] and bone metastasis [7], whereby temperatures >48C are utilised to kill the cancer cells. Additionally, using experimentally informed computational methods, we have predicted in a previous study that embedded osteocytes immediately experience the thermal elevations of the surrounding matrix in which they are entombed [8]. Temperature elevations trigger responses at the cellular level, such as cellular apoptosis and necrosis, which in turn lead to organ level reactions, the extent of which is dependent on the temperature itself and the duration of exposure [9,10,11]. Our recent study has shown that in vitro exposure of bone cells to temperatures exceeding 45C can trigger cellular responses, such as necrosis and apoptosis, depending on the extent and duration of thermal exposure and the phenotype of the cell [11]. Specifically, we showed that osteocyte-like cells are more resilient to heat-induced cellular death than osteoblast-like cells, whereby a large apoptotic response was observed at 12, 24 hours and 4 days after osteocyte-like MLO-Y4 cells were exposed to elevated temperatures ( 45C), and this apoptotic response occurred to a lesser extent in osteoblastlike MC3T3-E1 cells. When thermal elevation was minimised to 45C for 1 minute MLO-Y4 cells completely recovered by 4 days (as indicated by percentage necrosis, viability and population size), whereas osteoblast-like MC3T3-E1 cells could only withstand the same temperature for 30 seconds. Additionally, mild thermal elevations (42.544C) have been shown to stimulate bone remodelling, trabecular bone formation and increase cortical bone density of the rabbit femur after undergoing surgical trauma [12]. Direct exposure of osteoprogenitor cells to mild thermal elevations (3942.5C) for extended durations ( 1 hour) induced differentiation along the osteoblastic lineage and enhanced mineralised nodule formation in vitro [13]. Interestingly, we have recently shown that markers related to osteogenesis (alkaline phosphatase activity and calcium deposition) were up-regulated in Mesenchymal Stem Cells (MSCs) when directly exposed to clinically relevant elevated temperatures 47C for much shorter durations (30 seconds) in vitro [8,11]. It is known that soluble factors secreted by mechanically stimulated osteocytes (conditioned media) significantly up-regulated the expression of osteogenic genes osteopontin and Cox2 by MSCs in vitro [14]. Moreover, the percentage of alkaline phosphatase labelled surfaces and bone formation rate were significantly increased in human trabecular bone ex vivo in response to mechanical stimulation [15]. Surgically induced matrix damage and fatigue induced micro-damage to bone are well understood initiators of osteocyte apoptosis in vivo [16,17,18,19,20]. Most recently, it has been shown that osteocyte apoptosis directly results in an up-regulation of pro-osteoclastic signaling markers by nearby healthy cells, and thereby initiates the repair response [17,21,22]. Such studies provide strong evidence that site-specific osteocyte apoptosis is the underlying mechanism by which targeted removal of damaged bone tissue by osteoclastic bone resorption is initiated. Osteocytes have been identified as the major orchestrators of skeletal activity, sensing mechanical and chemical stimuli to regulate bone rem (...truncated)