| Summary: | 碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 101 === Bisphosphonates have been predominately applied in the treatment of osteolytic bone diseases, such as osteoporosis, Paget''s disease, multiple myeloma, and cancer-related metastatic bone lesions for the past several decades. Their clinical applications have alleviated the osteolytic symptoms, pain and fractures entailed by those diseases.
However, the implications of those drugs have led to new impacts on clinical dental treatments. Recently, an increasing number of clinical cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) have been reported, rendering the precautious measures in the treatments of dental extractions, periodontal surgery and dental implants necessary. Previous studies have shown that osteoclasts are the primary target cells and most vulnerable to bisphosphonates treatments. However, most of the experiments were confined to in vitro cell culture studies, simplicity of cell types in the experiments. On the other hand, the in vivo studies under physiological conditions are still elusive. Although clinical statistics and research in the cell biology field have gained some insights and have led to several hypotheses, the etiology of BRONJ remains to be explored in much more details.
To investigate the effects of bisphosphonate on osteoclasts and to unravel the in vivo mechanism underlying BRONJ, I employed a fin-regeneration model in an animal model--zebrafish. Zebrafish caudal fin rays were amputated and allowed to regenerate in the presence of alendronate, an aminobisphosphonate, at three doses. The growth tendencies of the fin ray bone were observed for 11 days after amputation. Osteoclasts were located by TRAP staining during the regeneration process.
In the first part of my study, I observe the dynamic appearance of osteoclasts after fin-amputation within 24 hours by TRAP staining without any drug treatment.
In the second part of the experiments, to clarify the impact of bisphosphonates on osteoclasts during the process of fin regeneration, I investigate the long-term effects of alendronate on osteoclast’s survival and distribution using the TRAP staining. Zebrafish were treated under three doses of administration of alendronate, 2.5×10-5 M, 5.0×10-5 M, 7.5×10-5 M, and 0 M as control. The samples were collected, stained, and observed at the first, 3rd, 5th, 7th, 9th and 11th day after amputation.
The results within 24 hours showed that by 18 hr, very few osteoclasts were scattered in the fin. In contrast, after 18 hr, many osteoclasts gradually gathered in the vicinity of amputated border sites evenly, located in the newly formed tissues as well as in the original tissues. After amputation, the dynamic distribution of osteoclasts within 24 hr implies that those cells may come from the circulation system and invade into the amputated border sites via extravasation, rather than from the local mesenchymal cells between fin hemirays.
The results of the long-term observation showed that the distribution of osteoclasts gradually diminished under the influence of alendronate administration; the appearance of TRAP+ osteoclasts was retarded and the duration of those cells on the regenerated fin was shortened. Those observations suggest that the survival and cellular activity of osteoclasts were impaired by alendronate. Contentiously, although the distribution and activity of osteoclasts were impeded by alendronate administration, the regeneration of fin ray (bone) was not ameliorated at high doses of alendronate administration. In contrast to previous studies in cell culture system, the results indicate that promiscuous cellular interaction exist during bone repairing, reflecting the etiological complication of BRONJ.
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