Background/Purpose: To facilitate an understanding of the biological factors involved in inflammation- mediated bone loss, in vitro systems and murine models of bone loss, such as endotoxin-induced calvarial erosion have been utilized.  However, challenges exist with in vivo model robustness, throughput and quantitation of bone erosions. Assessment of erosion is typically determined via micro-computed tomography (MicroCT) but quantitation has been hampered by mouse to mouse variability in bone morphology and lack of consistent robust erosion obtained by endotoxin alone. .   We set out to develop a robust, quantitative  in vivo endotoxin-induced calvarial erosion model that would be amenable for in vivo testing of potential therapeutic agents and would allow for gaining insight into processes involved in inflammatory bone loss.

Methods: Lipopolysaccharide (LPS) was injected with or without receptor activator of NF-kB ligand (RANKL), over the calvaria of mice.  On day 5 or 8, mice were euthanized and processed for MicroCT analysis.  Skulls were scanned at medium resolution, 70 kVp, 114 µA.  Scans were rendered into 3-D.  Optimal viewing angles were chosen for creation of TIFF images, which were then examined using Pax-IT imaging analysis software (MIS, Inc), using a 4-pass analysis to generate 4 areas of erosion representing each of the 4 calvarial plates.   The values obtained in the 4 areas of erosion were summed to provide a measure of total area of calvarial erosion.  For further validation aimed at demonstration of the quantitative nature of the model, in vivo testing of therapeutic agents known to play a role in osteoclastogenesis was implemented.

Results: Five-day dose-response studies using RANKL + LPS in B10.RIII mice resulted in severe erosive events, but dose-dependent relationships were not consistent.  We compared 5-day and 8-day models.   At Day 8, mice exhibited consistent and strong dose-dependent calvarial erosion with co-administration of 25 µg LPS and 10ug RANKL per mouse.  We determined inclusion of RANKL was necessary for optimal erosion.   Summating four 8-day studies using B10.RIII mice, co-administration of 25 µg LPS and 10 µg RANKL led to an average area of erosion of 4080 + 259 mm², compared to the naïve (untreated) average area of erosion of 506 + 40 mm².  The microCT/Pax-IT analysis method facilitated reproducible, quantitative results.  Importantly, therapeutic agents known to play a role in osteoclastogenesis demonstrated statistically significant reductions in bone erosion further emphasizing the quantitative nature of this model.

Conclusion: We developed a robust, quantitative endotoxin plus RANKL-induced calvarial bone erosion model through use of MicroCT and Pax-IT analysis.  This model and quantitation methodology allows for testing therapeutic agents to facilitate understanding of the biological factors and processes involved in bone erosion.  The ultimate goal is to impact identification of better therapeutic options for patients.

---

« Back to 2012 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/development-of-a-quantitative-model-of-endotoxin-induced-calvarial-bone-erosion-utilizing-micro-computed-tomography-and-pax-it-imaging-analysis-software/