Differential effects of biologic versus bisphosphonate inhibition of wear debris-induced osteolysis assessed by longitudinal micro-CT

Abstract

Aseptic loosening of total joint replacements is caused by wear debris-induced osteoclastic bone resorption, for which bisphosphonates (BPs) and RANK antagonists have been developed. Although BPs are effective in preventing metabolic bone loss, they are less effective for inflammatory bone loss. Because this difference has been attributed to the antiapoptotic inflammatory signals that protect osteoclasts from BP-induced apoptosis, but not RANK antagonists, we tested the hypothesis that osteoprotegerin (OPG) is more effective in preventing wear debris-induced osteolysis than zoledronic acid (ZA) or alendronate (Aln) in the murine calvaria model using in vivo micro-CT and traditional histology. Although micro-CT proved to be incompatible with titanium (Ti) particles, we were able to demonstrate a 3.2-fold increase in osteolytic volume over 10 days induced by polyethylene (PE) particles versus sham controls (0.49 +/- 0.23 mm(3) versus 0.15 +/- 0.067 mm(3); p < 0.01). Although OPG and high-dose ZA completely inhibited this PE-induced osteolysis (p < 0.001), pharmacological doses of ZA and Aln were less effective but still reached statistical significance (p < 0.05). Traditional histomorphometry of the sagital suture area of calvaria from both Ti and PE-treated mice confirmed the remarkable suppression of resorption by OPG (p < 0.001) versus the lack of effect by physiological BPs. The differences in drug effects on osteolysis were largely explained by the significant difference in osteoclast numbers observed between OPG versus BPs in both Ti- and PE-treated calvaria; and linear regression analyses that demonstrated a highly significant correlation between osteolysis volume and sagittal suture area versus osteoclast numbers (p < 0.001).

Figure 1. OPG vs. ZA inhibition of Ti-induced osteolysis

Mice (n=8) received sham or Ti surgeries to induce osteolysis over 10 days, and were given PBS, OPG or ZA (0.1mg/kg) treatment as indicated. Afterwards, calvaria were harvested for Orange/G and TRAP stained histology to quantify the SSA (A) and osteoclast numbers (B) as described in Materials and Methods. The data are presented as the mean +/− SD (* p<0.05, ** p<0.01, *** p<0.001 vs. PBS control with Tukey test). Representative Orange/G (C-F) and TRAP (G-M) stained histology are presented at 100X (C-J). High power images of the boxed regions in H and J are also shown at 400X (K-M). Of note are the large number of active osteoclasts in the PBS group (H & K), the complete absence of osteoclasts in the OPG group (I) and the presence of both active (L) and apoptotic (M) osteoclasts in the ZA groups.

Figure 2. Development of 3D Micro-CT quantification of PE-induced osteolysis

2D in vivo micro-CT images of calvaria 10 days after implantation of Ti and PE particles (A). Of note is the high signal produced by the Ti particles (arrow), which could not be accurately segmented away from the bone. In contrast, the absence of a Micro-CT signal from the PE particles allows for reconstruction of the bone volume from the day 0 and day 10 scans, and subtraction of the day 10-bone volume from the day 0-bone volume of a defined region of interest to quantify the volume of osteolysis (B).

Figure 3. OPG vs. BP inhibition of Ceridust-induced osteolysis assessed by longitudinal 3D micro-CT

Mice (n=8) received baseline in vivo micro-CT scans before sham or PE surgeries to induce osteolysis over 10 days, and were given PBS, OPG, low dose ZA, high dose ZA or Aln treatment as indicated. Afterwards, the mice received an in vivo micro-CT scan to quantify volumetric osteolysis as described in Figure 2. Representative reconstructed images of the calvaria and osteolytic volume from each group are shown (A). The osteolytic volumes from each group are presented as the mean +/− SD (* p<0.05, ** p<0.01, *** p<0.001 vs. PBS control with Tukey test) (B).

Figure 4. OPG vs. BP inhibition of Ceridust-induced osteolysis assessed by histology

After the day 10 in vivo micro-CT scans, the mice described in Figure 3 were sacrificed and their calvaria were harvested for Orange/G and TRAP stained histology to quantify the SSA (A) and osteoclast numbers (B) as described in Figure 1. The data are presented as the mean +/− SD (* p<0.05, ** p<0.01, *** p<0.001 vs. PBS control in Tukey test). Representative Orange/G (C-F) and TRAP (G-M) stained histology are presented at 100X (C-J). High power images of the boxed regions in H and J are also shown at 400X (K-M). Of note are the large number of active osteoclasts in the PBS group (H & K), the complete absence of osteoclasts in the OPG group (I) and the presence of both active (L) and apoptotic (M) osteoclasts in the low dose ZA group.

Figure 5. Differences in osteolysis between OPG vs. BP treated mice correlate with osteoclast number

Linear regression analyses were performed to assess the relationship between osteolysis determined by 3D micro-CT in Figure 3B (A) and 2D histomorphometry in Figure 4A (B), versus the number of osteoclasts in Figure 4B.