비스포스포네이트 관련 악골괴사- 세포 특이적 반응과 임상적 치료 결과 연구

Abstract

학위논문 (박사)-- 서울대학교 대학원 치의학대학원 치의과학과, 2017. 8. 서병무.Background and Purpose Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a challenging complication, the pathophysiology of which is not completely understood. The aims of this study were two folds: (1) to identify the risk factors associated with relapse or treatment failure after surgery for BRONJ in osteoporosis patients and (2) experimentally to investigate the effect of zoledronate on dental stem cells. Part I. Clinical study [Patients and methods] This was a retrospective cohort study of BRONJ in patients with osteoporosis who had undergone surgical procedures between 2004 and 2016 at the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital. The heterogeneous predictor variables were demographic (age and sex), anatomic (maxilla, mandible, maxilla plus mandible, affected location), clinical (disease stage, etiology, comorbidities, and history of intravenous bisphosphonate intake), time-related (conservative treatment before surgery, bisphosphonate treatment before BRONJ development, discontinuation of the drug before surgery, interval to final follow-up, and interval to reoperation in the case of relapse or treatment failure), and perioperative variables (type of anesthesia and type of surgical procedure). The primary outcome variable was relapse after surgery that required reoperation (yes vs. no). Descriptive and bivariate statistics were computed to assess the relationships among the study variables and the outcome. To determine risk factors, a survival analysis was conducted using the Cox model. [Results] The final sample consisted of 325 subjects with a median age of 75 years. Ninety-seven percent of the subjects were female. After surgery, 30% of the patients did not completely recuperate and received repeat surgeries. The time from the first surgery to reoperation ranged from 10 days to 5.6 years. Relapse or treatment failure occurred most often immediately after surgery. The type of surgical procedure and type of anesthesia were the most important factors for the treatment outcome. Drug holiday did not appear to influence the occurrence of relapse after surgery. Part II. In vitro study [Materials and methods] Bone marrow stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs) treated with various concentrations of zoledronate were investigated with regard to proliferation and differentiation capacity. The osteogenic differentiation of dental stem cells treated with zoledronate was evaluated by alizarin red S staining and alkaline phosphatase activity staining. RT-PCR was performed to compare MSX1, osteocalcin, collagen type I, and EF1a mRNA levels. Apoptosis was detected using FITC-Annexin V and PI staining assay. The dental stem cells were treated with bone morphogenetic protein (BMP)-2 to evaluate the possibility of overcoming the effects of zoledronate. [Results] High concentrations of zoledronate suppressed PDLSC and BMSC viability. The apoptosis of PDLSCs and BMSCs was triggered by both zoledronate and BMP-2. Higher concentrations of zoledronate triggered apoptotic activity in BMSCs, but additional BMP-2 treatment did not affect the apoptotic activity triggered by zoledronate. Moreover, zoledronate treatment significantly suppressed the formation of calcium deposits on PDLSCs and BMSCs regardless of BMP-2 treatment. BMP-2 treatment did not overcome the inhibitory effects of zoledronate on the osteogenic differentiation of PDLSCs and BMSCs. The MSX1 gene, highly expressed during craniofacial development, was significantly suppressed by 10 μM of zoledronate during the osteogenic differentiation of PDLSCs and BMSCs. Conclusions Surgical treatment of BRONJ in osteoporosis patients may benefit from more careful and extensive surgical procedures rather than curettage under local anesthesia. Zoledronate at high doses inhibited the proliferation, osteogenic differentiation, and MSX1 expression of dental stem cells. These findings indicate that zoledronate has negative effects on the regenerative capacity of dental stem cells, which may also explain the disease progression of BRONJ.Introduction Part I. Clinical study A. Patients and methods 1. Study design and sample 2. Study variables 2.1 Predictor variables 2.2 Outcome variable 3. Statistical analysis B. Results 1. Descriptive summary of study sample 2. Risk factors in the relapse/treatment failure group Part II. In vitro study A. Materials and methods 1. Human periodontal ligament stem cell (PDLSC) and human bone marrow stem cell (BMSC) primary culture 2. Proliferation assay 3. Apoptosis assay 4. Osteogenic differentiation 5. RT-PCR 6. Statistical analysis B. Results 1. Morphology of PDLSCs and BMSCs treated with zoledronate 2. Proliferation of PDLSCs and BMSCs treated with zoledronate and/or rhBMP-2 3. Apoptosis of PDLSCs and BMSCs treated with zoledronate and/or rhBMP-2 4. Effects of zoledronate and/or rhBMP-2 on osteogenic differentiation in PDLSCs and BMSCs 5. Changes in gene expression by zoledronate and/or BMP-2 treatment Discussion Conclusions References Tables Figures and Figure Legends Abstract in KoreanDocto