산업공정 리그닌을 이용한 약물 방출 조절형 나노입자 합성

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 산림과학부(환경재료과학전공), 2021. 2. 최인규.There has been growing interest in lignin as a promising source for drug delivery system preparation in recent years. The lignin-based nanoparticle has the potential to be preferably used because of its controllable intracellular uptake and stimuli-controlled drug-releasing ability. However, few research investigated the effect of structural characteristics of lignin on the synthesis of nanoparticles and its essential features such as size, colloidal stability, and biocompatibility. This research focuses on how the molecular structures of technical lignin (kraft and alkaline/soda lignin) affect the lignin nanoparticle properties. Based on these results, drug-encapsulated lignin nanoparticles were synthesized, and those drug-releasing efficiencies were investigated through evaluation of in vitro and in vivo biocompatibility. First, lignin fractions with different molecular weights (Mw 1,460 ~ 12,900), phenolic hydroxyl group (total 3.7 ~ 6.2 mmol g-1), and main internal linkages (β-O-4, β-β, and β-5) were obtained by performing a sequential solvent extraction of kraft lignin (KL). Subsequently, spherical lignin nanoparticles (KLNPs, i.d. 193 ~ 1,039 nm) were synthesized by the nanoprecipitation process. The low-molecular-weight KL fractions generated KLNPs with a relatively enlarged diameter but less size-distributed. Further analysis showed that molecular weight, total, and phenolic hydroxyl group content in the lignin highly correlated with nanoparticle size. Every KLNPs exhibited comparable and good colloidal stability (-44.5 ~ -29.1 mV). Cell viability and hemotoxicity assessments revealed higher and sustained biocompatibility of KLNPs even at a high concentration, at least for three days. Second, KL was utilized as a polymer for the drug-encapsulated nanoparticle (ELNP) synthesis. Successful drug encapsulation of ELNPs by nanoprecipitation was determined (max. 59% for coumarin 6 and max. 73% for doxorubicin (DOX)). Compared to KLNP, the size (i.d. 176 ~ 469 nm) and surface charge (-40.5 ~ -32.7 mV) of ELNP showed no remarkable change regardless of introducing the drug in the particle. The drug release profile of ELNPs was faster in the relatively acidic condition (over 70% within 0 ~ 4 h) than the neutral (less than 50% within 0 ~ 4 h). In vitro cytotoxicity assay revealed the drug release effect of ELNPs, delaying but not decreasing the anticancer activity. In addition, the hemocompatibility test, in vivo allergic reaction test, and tumor inhibition assays using mice tumors showed high ELNP biocompatibility and side effect reduction during the chemotherapy. Lastly, alkaline/soda lignin (AL) was sequentially solvent-extracted, or its phenolic hydroxyl groups were chemoselectively methylated. These lignins with the modified structure were handled to form nanoparticles (ALNP) to clarify the effect of the lignin structure on the physical properties of nanoparticles. Pure ALNPs were obtained from neat lignin, solvent-extracted fractions (ALNP-Fs, i.d. 414 ~ 1,214 nm), and methylated lignins (ALNP-Ms, i.d. 516 ~ 721 nm). Specifically, the size properties of ALNP-Ms showed a very high negative correlation (R2 = 0.95) with the phenolic hydroxyl group amount. It indicates that the phenolic hydroxyl groups in lignin are the main structures that affect the nucleation and/or condensation during the nanoprecipitation process. ALNPs exhibited high colloidal stability (-42.3 ~ -32.7 mV), and most of them also showed good in vitro biocompatibility.최근 리그닌을 약물 전달 시스템의 재료로 이용하는 연구가 주목받고 있다. 특히 리그닌으로 나노입자를 제조하는 연구는 약물 수송 및 방출 조절에 관한 장점을 부여할 수 있어 선호되는 추세이다. 하지만 그중 리그닌의 구조적 특성이 나노입자의 형성 과정이나 물성(크기, 콜로이드 안정성, 생체안전성)에 미치는 영향에 주목한 연구는 거의 없는 실정이다. 본 연구에서는 산업공정 부산물로 발생하는 리그닌 부산물의 구조적 특성이 리그닌 나노입자의 물성에 미치는 영향을 이해하고자 하였다. 해당 결과를 바탕으로 약물 저장 리그닌 나노입자를 합성하여 약물 방출 특성과 기대 효과를 생체외 및 생체내 조건에서의 생체안전성 실험을 통하여 구명하였다. 우선 산업공정 부산물인 kraft 리그닌을 순차적 용매 분획화하여 분자량, 수산기 함량, 분자간 주요 결합 함량이 각기 다른 5개 분획으로 분리하였다. 각 리그닌 분획으로부터 THF-리그닌 용액과 증류수의 치환에 의한 나노침전법을 통하여 나노입자를 획득하였으며 입자 크기는 최소 193 nm에서 최대 1,039 nm로 다양하게 나타났다. 리그닌 분자량이 작을수록, 페놀성 수산기 함량이 높을수록 크면서 균질한 분포를 보이는 입자가 형성되었으며 kraft 리그닌의 구조적 특성과 형성된 나노입자의 입자 크기와 분포는 리그닌 분자량과 수산기 함량, 특히 페놀성 수산기 함량에 영향을 받는 것으로 판단되었다. 나노입자는 대체로 깔끔한 구형으로 형성되었으며 증류수에 안정적인 콜로이드 상태로 분포하였다(-44.5 ~ -29.1 mV). 세포 생존율 및 혈구독성 평가 결과 리그닌 나노입자는 높은 농도 조건에서도 최소 3일간 높은 수준의 지속적인 세포안전성을 보이는 것을 확인하였다. 위 결과를 바탕으로 선택한 특정 리그닌 조건으로부터 약물 저장 나노입자를 합성하였으며 약물 방출 조절 효과를 입증하기 위한 실험을 수행하였다. 약물 저장 리그닌 나노입자는 coumarin 6에 대하여 최대 59%, 독소루비신에 대하여 최대 73%의 약물 저장 효율을 나타내었다. 약물 저장 리그닌 나노입자의 크기(176 ~ 469 nm)는 대조군과 비교하였을 때 약물 첨가에 큰 영향을 받지 않는 것으로 나타났다. 또한, 약물 저장 여부는 나노입자의 콜로이드 안정성에도 영향을 미치지 않았다. 약물 저장 리그닌 나노입자는 대체로 0 ~ 4시간 사이에 빠른 약물 방출 경향을 보였고 혈액과 동일한 pH 조건보다는 산성 조건에서의 약물 방출이 더 빠른 것으로 나타났다. 리그닌 나노입자는 생체외 혈구독성 실험과 생체내 알레르기 반응 실험에서 우수한 생체안전성을 나타냈으며, 생체외 세포독성 실험과 생체내 항암효과 검증 실험에서 약물 방출 조절 및 부작용 경감에 의한 사망률 감소 효과를 나타냈다. Alkaline/soda 리그닌의 경우 순차적 용매 분획화와 별도로 페놀성 수산기의 메틸화 반응을 수행하여 분자량의 변화가 상대적으로 적으면서 페놀성 수산기의 함량이 각기 다른 4개 그룹을 추가로 획득하였다. 구조적 변형을 거친 해당 리그닌 그룹으로부터 나노입자를 획득하여 그 물성을 분석한 결과, 분획화 리그닌 유래 나노입자는 kraft 리그닌에서와 유사한 크기 분포 경향을 보였다. 반면, 메틸화 리그닌 유래 나노입자는 페놀성 수산기가 감소함에 따라 크기가 감소하는(721 ~ 516 nm) 뚜렷한 경향을 보였다. 모든 조건에서 나노입자의 콜로이드 안정성은 높게 유지되었으며 생체안전성 역시 대부분 농도 및 시간 조건에서 안전성을 나타냈다. 본 연구에서는 펄프 및 바이오에탄올 생산공정에서 부산물로 발생하는 리그닌의 고부가가치 활용방안으로 약물 방출 조절 나노입자 합성을 제시하였다. 리그닌의 구조적 특성과 나노입자 물성의 상관성을 구명하여 본 연구에서 다루지 않은 다른 리그닌을 이용한 나노입자 제조 및 물성 제어에 있어서도 단초를 제공하였으며, 생체외 및 동물 실험을 통한 리그닌 나노입자의 약물 방출 조절 및 부작용 경감 효과를 검증하여 약물 전달체 원료로써 실제 적용 가능성을 제시하였다.Contents Chapter 1 Introduction 1 1. Background 2 1.1. The concept of lignocellulosic biorefinery 2 1.2. Lignin production from pulping and sugar-based biorefinery 3 1.3. Understanding the lignin structure for further application 6 1.4. Polymeric nanoparticle for biomedical use 8 2. Objectives 12 3. Literature review 15 3.1. Valorization of lignin macromolecules 15 3.1.1. Isolation of lignin from biomass 15 3.1.2. Determination of lignin structure 19 3.1.3. Lignin fractionation 24 3.1.4. Structural functionalization of lignin 28 3.2. Nano-sized carriers for drug delivery 38 3.2.1. Strategies for drug delivery 38 3.2.2. Nanoparticle synthesis 44 3.2.3. Strategies for controlled drug release 48 Chapter 2 Synthesis of pure nanoparticles with solvent-fractionated kraft lignin and evaluation of their biocompatibility 51 1. Introduction 52 2. Materials and methods 54 2.1. Materials 54 2.2. Lignin fractionation 56 2.3. Characterization of fractionated lignin 58 2.4. Synthesis of lignin nanoparticle 62 2.4.1. Pre-dialysis concentration 62 2.4.2. Lignin fractions 64 2.5. Characterization of lignin nanoparticle 65 2.6. Biocompatibility tests 66 2.6.1. CCK-8 assay 66 2.6.2. Hemolysis assay 68 2.7. Statistical analysis 69 3. Results and discussion 70 3.1. Characteristics of lignin fractions 70 3.2. Particle size and morphology 80 3.3. Particle surface charge 89 3.4. Storage stability of the particle in pure water 90 3.5. In vitro cell viability 93 3.6. Hemocompatibility 98 4. Conclusion 100 Chapter 3 In vivo and in vitro evaluation of drug-encapsulated lignin nanoparticle 101 1. Introduction 102 2. Materials and methods 106 2.1. Materials 106 2.2. Animals and ethics 108 2.3. Synthesis of encapsulated lignin nanoparticle 109 2.3.1. Coumarin 6-encapsulated nanoparticle 109 2.3.2. DOX-encapsulated nanoparticle 110 2.4. Characterization of encapsulated nanoparticle 111 2.4.1. Determination of physical properties of nanoparticle 111 2.4.2. Drug loading 112 2.4.3. In vitro release study 113 2.5. Biocompatibility tests 114 2.5.1. CCK-8 assay 114 2.5.2. Hemolysis assay 115 2.5.3. In vivo detection of an allergic reaction 116 2.6. In vivo anticancer efficacy 117 2.7. Statistical analysis 118 3. Results and discussion 119 3.1. Particle size and encapsulation efficiency 119 3.1.1. Coumarin 6-encapsulated nanoparticle 119 3.1.2. DOX-encapsulated nanoparticle 124 3.2. Particle surface charge 128 3.3. In vitro drug release 130 3.4. In vitro cytotoxicity 135 3.5. Hemocompatibility 138 3.6. Hypersensitivity test 140 3.7. In vivo antitumor efficacy 142 4. Conclusion 145 Chapter 4 Effect of chemoselective methylation of the phenolic hydroxyl group on lignin nanoparticle synthesis 147 1. Introduction 148 2. Materials and methods 150 2.1. Materials 150 2.2. Functionalization of lignin 152 2.3. Characterization of lignin 154 2.4. Synthesis of lignin nanoparticle 156 2.4.1. Pre-dialysis concentration 156 2.4.2. Functionalized lignin 158 2.5. Characterization of lignin nanoparticle 159 2.6. Biocompatibility test 160 2.7. Statistical analysis 161 3. Results and discussion 162 3.1. Characteristics of functionalized lignin 162 3.1.1. Lignin fractions 162 3.1.2. Lignin methylation 174 3.2. Effect of lignin characteristics on nanoparticle size 178 3.2.1. Effect of solvent fractionation 178 3.2.2. Effect of the hydroxyl group 186 3.3. Particle surface charge 190 3.4. Storage stability of nanoparticle 192 3.5. In vitro cell viability 195 4. Conclusion 198 Chapter 5 Conclusion 199 References 203 초록 225Docto

Therapeutic Effects of HIF-1α on Bone Formation around Implants in Diabetic Mice Using Cell-Penetrating DNA-Binding Protein

Patients with uncontrolled diabetes are susceptible to implant failure due to impaired bone metabolism. Hypoxia-inducible factor 1α (HIF-1α), a transcription factor that is up-regulated in response to reduced oxygen during bone repair, is known to mediate angiogenesis and osteogenesis. However, its function is inhibited under hyperglycemic conditions in diabetic patients. This study thus evaluates the effects of exogenous HIF-1α on bone formation around implants by applying HIF-1α to diabetic mice and normal mice via a protein transduction domain (PTD)-mediated DNA delivery system. Implants were placed in the both femurs of diabetic and normal mice. HIF-1α and placebo gels were injected to implant sites of the right and left femurs, respectively. We found that bone-to-implant contact (BIC) and bone volume (BV) were significantly greater in the HIF-1α treated group than placebo in diabetic mice (p < 0.05). Bioinformatic analysis showed that diabetic mice had 216 differentially expressed genes (DEGs) and 21 target genes. Among the target genes, NOS2, GPNMB, CCL2, CCL5, CXCL16, and TRIM63 were found to be associated with bone formation. Based on these results, we conclude that local administration of HIF-1α via PTD may boost bone formation around the implant and induce gene expression more favorable to bone formation in diabetic mice.ope

Hormone Therapy in the Era of the COVID-19 Pandemic: A Review

The coronavirus disease 2019 (COVID-19) pandemic has impacted the medical, social, and reproductive health of millions of people since its outbreak. The causative virus transmits, reproduces, and manifests through the respiratory tract. COVID-19 can invade any system of the body, including the cardiovascular and endocrine systems, through a secondary immune response. In particular, because the fatality rate is high in those over the age of 50 years, special attention is required during the medical care of this population. However, considering the benefit of therapy and the risk of COVID-19, high-quality evidence regarding individualized management in relation to hormone therapy is still insufficient in the field of gynecology. Furthermore, this review aims to serve as a reference for clinical application by analyzing and summarizing the results of studies reported to date regarding female hormone therapy in the context of the COVID-19 pandemic.ope

The implication of holocytochrome c synthase mutation in Korean familial hypoplastic amelogenesis imperfecta

Objectives: This study aimed to comprehensively characterise genetic variants of amelogenesis imperfecta in a single Korean family through whole-exome sequencing and bioinformatics analysis. Material and methods: Thirty-one individuals of a Korean family, 9 of whom were affected and 22 unaffected by amelogenesis imperfecta, were enrolled. Whole-exome sequencing was performed on 12 saliva samples, including samples from 8 affected and 4 unaffected individuals. The possible candidate genes associated with the disease were screened by segregation analysis and variant filtering. In silico mutation impact analysis was then performed on the filtered variants based on sequence conservation and protein structure. Results: Whole-exome sequencing data revealed an X-linked dominant, heterozygous genomic missense mutation in the mitochondrial gene holocytochrome c synthase (HCCS). We also found that HCCS is potentially related to the role of mitochondria in amelogenesis. The HCCS variant was expected to be deleterious in both evolution-based and large population-based analyses. Further, the variant was predicted to have a negative effect on catalytic function of HCCS by in silico analysis of protein structure. In addition, HCCS had significant association with amelogenesis in literature mining analysis. Conclusions: These findings suggest new evidence for the relationship between amelogenesis and mitochondria function, which could be implicated in the pathogenesis of amelogenesis imperfecta. Clinical relevance: The discovery of HCCS mutations and a deeper understanding of the pathogenesis of amelogenesis imperfecta could lead to finding solutions for the fundamental treatment of this disease. Furthermore, it enables dental practitioners to establish predictable prosthetic treatment plans at an early stage by early detection of amelogenesis imperfecta through personalised medicine.ope

18F]Mefway 양전자방출단층촬영술로 측정한 갑상선기능저하증이 쥐 뇌의 세로토닌 1A 수용체에 미치는 영향

Dept. of Medicine/박사This study investigated the effects of hypothyroidism on the brain serotonin (5-HT) 1A receptor using animal disease models and 4-(trans-[18F]fluoranylmethyl)-N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexane-1-carboxamide ([18F]Mefway) positron emission tomography (PET) imaging. Five surgically thyroidectomized male Sprague-Dawley rats were assigned to the hypothyroidism group, and five sham-operated male Sprague-Dawley rats were assigned to the control group. Hypothyroid status was confirmed by thyroid function tests. After anesthesia with 2.0% isoflurane in oxygen, fluconazole was infused at a constant rate for one hour to prevent defluorination of the radioligand. Then [18F]Mefway of 8.6–11.1 MBq was administered at a rate of 1 ml/min and dynamic PET scans were performed over the course of 120 min. PET data were reconstructed in user-defined time frames using a two-dimensional ordered-subset expectation maximization algorithm. All PET data were spatially normalized to T2-weighted magnetic resonance imaging templates, and then time-activity curves of the hippocampus, septum, and cerebellum were extracted using predefined volume of interest templates. Non-displaceable binding values in the hippocampus and septum were calculated using a multilinear reference tissue model, with the cerebellum as the reference tissue, and a ligand-specific parametric map was constructed. Time-activity curves revealed that the hippocampal and septal uptakes in the hypothyroidism group were 25–52% higher than those in the control group, and non-displaceable binding potentials in the same regions of thyroidectomized rat brains were about 30% higher than those in controls. In conclusion, hypothyroidism increased the density of postsynaptic 5-HT1A receptors in the hippocampus and septum. Upregulation of postsynaptic 5-HT1A receptors may be responsible for the increased radioligand uptake as an early response to the reduced synaptic concentration of 5-HT caused by hypothyroidism. 이 연구는 동물 질병 모델 및 4-(trans-[18F]fluoranylmethyl)-N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-ylcyclohexane-1-carboxamide ([18F]Mefway) 양전자방출단층촬영술(PET)을 사용하여 갑상선기능저하증이 뇌의 세로토닌 1A 수용체에 미치는 영향을 연구하였다. 갑상선절제술을 받은 Sprague-Dawley 수컷 쥐 5마리를 갑상선기능저하증 집단에 배정하였고, 모의 수술(sham operation)을 받은 Sprague-Dawley 수컷 쥐 5마리를 대조군으로 설정하였다. 갑상선기능저하 상태는 갑상선 기능 검사로 확인하였다. 산소에 2%의 아이소플루레인을 혼합하여 마취를 시킨 후, [18F]Mefway의 탈불소화를 방지하기 위하여, 1시간 동안 플루코나졸을 투여하였다. 그 다음 8.6–11.1 MBq의 [18F]Mefway를 1 ml/min 속도로 투여한 후, PET 영상을 120분간 연속적으로 획득하였다. PET 영상은 2차원 정칙화된 기댓값 최대화 알고리즘(ordered subsets expectation maximization algorithm)을 이용하여 재구성하였다. 모든 PET 영상은 T2 강조 자기공명영상 틀(template)에 공간정규화 한 후 사전 정의된 관심영역 틀을 이용하여 해마, 사이막(septum), 소뇌의 시간방사능곡선을 추출하였다. 해마와 사이막의 수용체 결합능(non-displaceable binding potential)은 소뇌를 참조조직으로 삼아 다중선형 참조조직 모형(multilinear reference tissue model)을 통하여 계산하였고 리간드-특정 모수지도(parametric map)를 작성하였다. 시간방사능곡선에서 갑상선기능저하증 집단의 해마와 사이막 섭취는 대조군보다 25-52% 더 많았고 이 부위의 결합능은 갑상선절제술을 받은 쥐에서 대조군에 비하여 30% 가량 더 높았다. 결론적으로, 갑상선기능저하증은 해마와 사이막의 시냅스후 세로토닌 1A 수용체 밀도를 증가시켰다. 이는 갑상선기능저하증으로 인해 시냅스의 세로토닌 농도가 감소하고 이에 따른 초기 반응으로써 시냅스후 세로토닌 1A 수용체가 상향조절 되었기 때문일 것이다.ope

Experimental Investigation of Reverbed Signal Due to Microbubbles within Ship Wake

Microbubbles are created in copious quantities in the wakes of ocean vessels and, because of their large acoustical cross sections, are responsible for the scattering acoustical signature of the wake. The acoustic characteristics of the suspended microbubbles can be used to detect and track the ship wake. In this thesis, we show that the experimental result performed at Korea Maritime University. It was calculated the volume scattering strength via experimental data. We can investigate the motion and distribution of bubble plume within ship wake via volume scattering strength.목차 Ⅰ 그림 목차 Ⅱ 표 목차 Ⅳ Abstract Ⅴ 1. 서 론 1 1.1. 연구의 목적 및 필요성 1 1.2. 연구의 내용 및 방법 3 1.3. 논문의 구성 4 2. 항적에 의한 음향산란특성 5 2.1. 체적산란강도 5 2.2. Wavelet 변환에 의한 난류 유동 특성 10 3. 항적에 의한 음향산란 해상실험 13 3.1. 실험환경 13 3.2. 실험 장비의 구성 16 3.3. 실험조건 및 방법 17 4. 실험결과 분석 19 4.1. 체적산란강도 19 4.1.1. 분석방법 19 4.1.2. 분석결과 23 4.2. Wavelet 변환에 의한 난류 유동 특성 39 4.2.1. 분석방법 39 4.2.2. 분석결과 41 5. 결 론 47 참고 문헌 4

Difficult intubation: lessons learned from the courts of South Korea

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Single Nucleotide Polymorphisms and Haplotypes in Vascular Endothelial Growth Factor Gene and Residual Ridge Resorption of Mandible in Korean Population

The tooth extraction is a routine surgical procedure in the dental treatment where the healing process results in a saddle-shaped residual ridge in the edentulous jaw. There are substantial differences among individuals in the end result. In some cases, there is excessive bone atrophy, which complicates the dental restorative treatment. The alveolar ridge receives the mechanical load continuously from the periodontal ligament connected to the teeth and it diminishes dramatically as a consequence of dental extraction; thus it is believed the continuing pattern of the alveolar bone resorption is related to this change. The reduced partial pressure of oxygen is the most prominent event from the reduced mechanical load. Vascular Endothelial Growth Factor (VEGF), regulated by HIF-1, reported close association with angiogenesis and bone turn over, where partial oxygen pressure has changed. Therefore the genetic association between Single Nucleotide Polymorphsim (SNP) of VEGF gene and RRR was investigated. 120 subjects (70.93 ± 9.28 years) which were treated at Dental clinic of Yonsei University with edentulous mandible were recruited. Mandibular bone height was measured following the protocol of the American College of Prosthodontists. Three variants, rs1570360, rs25648, and rs3025039 in VEGF from previous study, were used as tag-SNPs and genotyping for the study. Student’s t-test and ANOVA were used for statistical analysis. There was a notable association with rs1570360 (P = 0.051) in dominant group and haplotype A-C-C showed a statistically significant association with RRR in dominant group (P = 0.042). Results of this study may be useful in developing novel genetic diagnostic tests and identifying Koreans susceptible to developing severe RRR after dental extraction.ope

Therapeutic use of mesenchymal stem cell-derived extracellular vesicles in acute lung injury

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Complications related to cantilever lengths in implant-supported single crowns

Prosthesis using implant has become a popular modality to treat edentulous patients for the last 35 years. The progress in surface treatment reduces the failure rate and provides better functional and esthetic restorations in dentistry. The earliest form of implant prosthesis was the splinted-fixed type. After full arch and partial edentulous prosthesis began to demonstrate s stable clinical successes rate, single crowns started to be restored with implants.When the implant supported single crown is located most distally in the arch, special biomechanical factors should be considered. Especially when the implant is placed in a more distal position, there will be exposure to tremendous lateral forces resulting in possible complications. The purpose of this study is to analyze the relationship between complications and distance from adjacent teeth to the implant, and to identify the types and frequency of complications. A total of 115 patients charts were investigated randomly from the implant clinic at Ewha WomansUniversity Hospital and Yonsei University Dental Hospital. The patients received an implant supported single crown from 1996 to 2007 and annual afterwards. Patients were grouped according to the presence or lack of complications and the relationship of tooth to implant distance and complications was investigated. Age, sex, and abutment connection type were also analyzed for relationship with complications. The measured data from the two groups were analyzed statistically with SAS version 9.1 (SAS Inc., USA). There were significant statistical differences using logistic analysis (p<0.05) between the tooth to implant distance and complications. The odds ratio was 2.1 and cut off value from maximum value of specificity and sensitivity was 3.39 mm. SPSS 12.0(SPSS Inc. Chicago, Illinois. USA)ope