Thermally induced osteocyte damage initiates a remodelling signaling cascade

Abstract

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 (47°C 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 47°C 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.

Fig 1. Heat-treatment induces damage responses in MLO-Y4 cells.

Phalloidin stained actin filaments (red) and DAPI stained nucleus (blue) of MLO-Y4 cells heat-treated to (A) 37°C (control) and (B) 47°C for 1 minute demonstrating membrane condensation (white arrows) and rounded cell bodies detaching (orange arrows). Scale bar = 32μm. (C) Flow cytometry quantification of necrotic, apoptotic and healthy cell populations 24 hours after heat-treatment. * indicating statistical difference in the number of viable, apoptotic and necrotic cells compared to the 37°C (control) (p ≤ 0.05).

Fig 2. Heat-treatment of MLO-Y4 cells causes a down-regulation in the Rankl/Opg ratio.

A) Rankl, B) Opg and C) Rankl/Opg gene expression by heat-treated (HT) MLO-Y4 cells to 47°C compared to the 37°C control group at 1, 3 and 7 days after heat-treatment (p≤0.05).

Fig 3. Heat-treatment of MLO-Y4 cells causes a late stage up-regulation in Cox2 expression.

Cox2 gene expression of heat-treated (HT) MLO-Y4 cells (47°C) compared to the 37°C control group at, 1, 3 and 7 days after heat-treatment (p≤0.05).

Fig 4. Co-culture of MLO-Y4 cells with heat-treated MLO-Y4s causes an initial up-regulation in the Rankl/Opg ratio.

A) Rankl, B) Opg and C) Rankl/Opg gene expression by MLO-Y4 cells co-cultured (ccMLO-Y4s) with MLO-Y4 cells heat-treated to 47°C compared to the 37°C control groups at 1, 3 and 7 days after heat-treatment (p≤0.05).

Fig 5. Co-culture of MLO-Y4 cells with heat-treated MLO-Y4s causes a late stage up-regulation in Cox2 expression.

Cox2 gene expression by MLO-Y4 cells co-cultured (ccMLO-Y4s) with MLO-Y4 cells that were heat-treated to 47°C compared to the 37°C control group at 1, 3 and 7 days after heat-treatment (p≤0.05).

Fig 6. Co-culture of MLO-Y4 cells with heat-treated MLO-Y4s increases Alkaline Phosphatase Expression.

Alkaline phosphatase activity of pre-osteoblastic MSCs 0, 4, 7, 14 and 21 days after their addition to MLO-Y4s heat-treated to 47°C, compared to the 37°C control (p≤0.05).

Fig 7. Co-culture of MLO-Y4 cells with heat-treated MLO-Y4s increases Calcium Deposition.

Calcium deposition of pre-osteoblastic MSCs 0, 4, 7, 14 and 21 days after their addition to MLO-Y4s heat-treated to 47°C, compared to the control 37°C (p≤0.05).