Developing a zebrafish model of bisphosphonate induced osteonecrosis of jaw to define cellular and molecular mechanisms

• Main Author: Ethiraj, Lalith Prabha
• Other Authors: Tom James Carney
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Medicine
• Online Access: https://hdl.handle.net/10356/168636
• Institution: Nanyang Technological University

Bisphosphonates are critical drugs of the orthopaedic clinic, which act by inhibiting osteoclast resorptive activity and thus promote bone density. They are commonly used for treating Osteogenesis Imperfecta (OI), Paget’s disease, osteoporosis, and bone metastasis. In particular, the global rise in the incidence of the latter two diseases has led to significantly expanded bisphosphonate use in recent years. This has seen a concomitant increase in the incidence of Bisphosphonate Related Osteonecrosis of Jaw (BRONJ), a rare but potentially serious side effect of bisphosphonate use. Patients affected by BRONJ show paradoxical necrotic lesions in the jaw and loss of teeth with a considerable reduction in quality of life. The rarity of BRONJ limits the power of human studies, and the underlying mechanism remains unclear. Treatment options are extremely limited and mostly surgical. This necessitates the need for animal models to determine the cellular and molecular mechanisms involved. Current mammalian models of BRONJ require surgical removal of teeth to instigate ONJ, limiting throughput, and their current clinical translational impact is questionable. In previous studies, we demonstrated bisphosphonate efficacy in zebrafish OI and fracture models. I have developed colorimetric and fluorescent Tartrate Resistant Acid Phosphatase (TRAP) staining approaches to assess and quantify osteoclast activity in the fracture model and on the tooth-bearing ceratobranchial bone. This allowed me to demonstrate a high spontaneous osteoclastic activity around the base of the zebrafish pharyngeal teeth and its suppression with bisphosphonate treatment. Surprisingly, I also noted necrotic lesions in the jaw of all adult zebrafish following extended treatment with bisphosphonate. This led to teeth-bone disjunction and was reminiscent of the BRONJ seen in the clinic. This establishes zebrafish as a robust and accessible model of BRONJ. There is convincing evidence of an immunological contribution to BRONJ progression. Using the newly established zebrafish BRONJ model, I assayed the innate immune response through a combination of fluorescent dyes and transgenic lines. A biphasic immune response was observed. There was an initial infiltration of neutrophils which ablated after 3 weeks, and activated macrophages were observed pooling at the teeth bone junction at later stages. Concomitant with this was an early downregulation of NF-κB signalling, followed by marked NF-κB signalling upregulation at later time points. I tested if the osteoclast/macrophage lineage was necessary for BRONJ. Genetic ablation of these cells using the csf1ra mutant entirely abolished BRONJ onset following alendronate treatment. Furthermore, I showed 9 pharmacological inhibition of NF-κB signalling was fully protective against BRONJ in zebrafish. In sum, I developed a robust zebrafish model of BRONJ and used it to generate preliminary insights into immune contribution to the pathology and finally identified a signalling pathway that can be targeted for treatment or prevention. Future uses of this model is to identify, describe and ameliorate BRONJ in the clinic, are discussed.