A new insight into the mechanism of bisphosphonate-related osteonecrosis of the jaw using zebrafish as a model
Date Issued
2010
Date
2010
Author(s)
Wu, Chun-Hsieh
Abstract
Bisphosphonates, analogues of endogenous pyrophosphate which binds to hydroxyapatite in bone, are synthetic drugs for treating osteoporosis. Endogenous pyrophosphate found in serum and urine is unstable and easily metabolized. In contrast, bisphosphonates have been developed as drugs due to their stability and affinity to bone, conferring the inhibition of bone-resorption probably mediated by impeding osteoclast function. To date, they have been broadly employed in medication for the patients with osteoporosis and metastatic osteolytic cancers, which betrays their impact on clinical therapies.
However, recent clinical reports have implicated these drugs in adverse effects on bone and wound healing. The first report about “bisphosphonate-related osteonecrosis of the jaw”(BRONJ) was published in 2003. Consecutively, many correlated cases have been identified. These contentious clinical phenomena begin to overwhelm and baffle the dentists as well as other medical doctors who have encountered the patients using bisphosphonates. The underlying detailed mechanism of BRONJ still remains elusive. The prevalent application of these drugs and its implication in dental surgeries and cancer therapies suggest this is an important compelling issue for medical research.
To date, there is no related research of BRONJ using zebrafish as a model. The teleost zebrafish (Danio rerio, osteichthye), a vertebrate animal model, has been extensively applied in bio-medical research, including osteogenesis. The regeneration of zebrafish caudal fin is similar to the regeneration of human intramembranous bone. The bone and cartilage of the fish can be easily visualized by staining, facilitating the observation of their morphologies. In this study, I utilize zebrafish to analyze the effects of bisphosphonate on bone re-growth for quantitative and qualitative analyses, dissect the etiology of BRONJ and unravel the underlying molecular mechanism by in vivo evidences.
At first I analyzed the morphologies of zebrafish embryos after treatment with alendronate. I observed the development of larvae cartilage by alcian blue staining and early facial and spinal bone mineralization(day 5~day 14) by calcein staining. In adult zebrafish, I observed the morphology of fin regeneration after amputation by alizarin red and calcein (in vivo) staining. Analysis of cell apoptosis was done by acridine orange and I perform in situ hybridization to detect early and late gene markers of osteoblast and osteoclast to unveil the gene expression patterns.
My results show that alendronate has no effect on larva cartilage development and early bone mineralization. But it has a dose-dependent effect on bone mineralization of adult fin regeneration. Low dose augments this process, while higher dose impedes it. Futhermore, it seems that this drug exerts cytotoxicity on the cells encompassing bone matrix, resulting in cell apoptosis. I found by in situ hybridization that low dose of alendronate augments the expression of Runx2, an early gene marker of osteoblast and higher dose suppress its expression. In conclusion: The results suggest the main cause of BRONJ is that alendronate is a bone hook and cumulatively bound in bone. When wound formation, the local bone turnover rate increases rapidly and releases large quantity of alendronate, which causes the death of cells surrounding fin rays and impedes the wound healing.
Keywords: bisphosphonate, osteonecrosis, zebrafish, bone
Subjects
bisphosphonate
osteonecrosis
zebrafish
bone
SDGs
Type
thesis
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