The effects of bisphosphonate on differentiation and distribution of osteoclast using zebrafish as an in vivo animal model

• Main Authors: Chia-Yuan Hu, 胡家源
• Other Authors: Jou-Song Wang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/59790776556097508000

碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 102 === Objectives： Bisphosphonates began to be prescribed in the 1980s, often used in osteoporosis, bone metastasis of malignant tumors, multiple myeloma. Over 200 million oral and nearly three million intravenous prescriptions were made until 2006. However, 36 cases of osteonecrosis of the jaw was first reported in 2003 by the American Oral and Maxillofacial Surgeons, Marx. Ten thousands cases was proposed later. People started to pay attention to the side effect of this drug. Because this drug delayed wound healing of the jaw bone, all dentoalveolar surgeries were involved, such as tooth extraction, root planing, periodontal surgery, or implant. Dentists pay special attention to this topic and phenomenon. Upon osteonecrosis of the jaw occuring, it takes long time to wait for wound healing. Moreover, reconstruction surgery is difficult and non-invasive treatment option was usually chosen. Thus, we would like to unveil the etiology of BRONJ, and hope to find the preventive evaluation and treatment methods. Various causative pathologies of bisphosphonate-related osteonecrosis of the jaw have been proposed. Inhibition of osteoclast function is generally considered to be the main mechanism, leading to cell apoptosis and defect in bone remodeling, but direct mechanism of action was unknown. Therefore, my research goal was to explore the effects of bisphosphonate on differentiation and distribution of osteoclast. Materials and Methods： Owing to the similar process between fin regeneration of zebrafish and intramembranous ossification bone growth of humans, we use zebrafish as an animal model to explore the effects of bisphosphonates on osteoctlast. Zebrafish were selected from two groups, one wild type group was revealed by TRAP staining (Tartrate-resistant acid phosphatase, TRAP) to mark osteoclasts; the other group is CTSK transgenic zebrafish by human osteoclast gene promoter encoding green fluorescent protein(GFP). The stain and fluorescence help us to observe the effect of bisphosphonates on osteoclasts in these fish. First of all, we amputate the zebrafish tail fin in half, and all fishes were divided into three groups according to breeding condition by concentration of Alendronate: the control group, the low concentration (2.5 × 10-5M) and the high concentration (7.5 × 10-5M) of Alendronate. On day 3 and day 5 after fin amputation, we collected the regenerated fin and revealed with TRAP staining. These specimens were &;#64257;xed overnight at 4 °C in 4% paraformaldehyde (PFA), dehydrated by ethanol, and embedded in resin (Technovit 9100, Kulzer Heraeus) according to the manufacturer''s instructions. Sections were cut at 7 μm and toluidine blue as counter stain. Then we observe the histology by cross-section slide, including the morphology of osteoclasts, the distribution of osteoclasts, to see if their differentiation or bone resorptive ability were suppressed by bisphosphonates. Also, we compared the extent of suppression by different drug concentration. Furthermore, these findings could help us understand the inhibited osteoclasts function affected by bisphosphonates. Finally, we compare the group of TRAP and CTSK transgenic zebrafish from gross view, expecting to find the similarities and differences of the distribution and origin of osteoclasts. Results： The results showed that multinucleated osteoclasts with rounded morphology increased after bisphosphonate use, their differentiation might be inhibited and resulted in impaired bone resorption. In addition, the TRAP stain of osteoclasts showed and the length of the regenerated tail were both decreased with increasing drug concentration, suggesting that bisphosphonates could cause the apoptosis of osteoclasts, supressed regeneration, and delayed wound healing. TRAP-marked osteoclasts were larger in number and distributed mostly at the cutting line and the area nearby the end of regenerated fin. Whereas the CTSK-specific osteoclasts were less revealed by TRAP stain and evenly-distributed over the regenerated fin. These results indicated that the origin of TRAP-marked osteoclasts was possible from the precursor cells in blood circulatory system, while CTSK-marked osteoclasts were probably derived from the mesenchymal cells of the fin rays. Conclusion： This experiment showed that osteoclast differentiation may be suppressed under the use of bisphosphonates, leading to increased cell apoptosis. Defects in wound healing, bone repair and regeneration may result in BRONJ.