A method for determining the rare elements in bones and similar materials includes [eight] steps. / Master of Science

Yes / The suspected scaphoid fracture remains a diagnostic conundrum with over-treatment a common risk-averse strategy. Cross-sectional imaging remains the gold standard with MRI recommended but CT used by some because of easier access or limited MRI availability. The aim of this feasibility study was to evaluate whether cone beam computed tomography (CBCT) could support early diagnosis, or exclusion, of scaphoid fractures. Patients with a suspected scaphoid were recruited fracture between March and July 2020. All underwent a 4-view X-ray. If this examination was normal, they were immediately referred for a CBCT scan of the wrist. Those with a normal scan were discharged to research follow-up at 2 and 6-weeks. 68 participants were recruited, 55 had a normal or equivocal X-ray and underwent CBCT. Nine additional radiocarpal fractures (16.2%) were demonstrated on CBCT, the remainder were discharged to research follow-up. Based on the 2-week and 6-week follow up three patients (4.4%) were referred for MRI to investigate persistent symptoms with no bony injuries identified. CBCT scans enabled a rapid pathway for the diagnosis or exclusion of scaphoid fractures, identifying other fractures and facilitating early treatment. The rapid pathway also enabled those with no bony injury to start rehabilitation, suggesting that patients can be safely discharged with safety-net advice following a CBCT scan.

「甲骨塗辭」乃指在甲骨刻辭筆畫中塗以朱、墨，以往學者將之稱為「塗朱/墨」或「填朱/墨」，這類刻辭在刻寫完成之後，以朱砂或墨填入刻痕。其中，塗朱甲骨色彩鮮明奪目，很早就受到學者注意，其目的有美觀、宗教意涵、重要訊息的不同說法。然而，受限於甲骨著錄多為黑白拓本形式、早期彩色印刷不發達，目前甲骨學界尚無學者對塗辭做出專門且深入的研究。所幸，近年來出版之甲骨著錄附有彩色相片，部分甲骨收藏單位架設數位資料庫，如中研院史語所，所能蒐羅之資料備於完善，為甲骨塗辭研究奠定基礎。 本文以「塗朱甲骨」作為主要討論對象，試圖將辭例或形狀完整的塗朱卜辭進行分類。首先於第二章探討塗朱甲骨的年代以及塗朱甲骨的幾項特徵，包含：牛胛骨骨面塗朱卜辭、犯兆塗朱卜辭、塗朱記事刻辭。第三章及第四章則以塗朱甲骨的形態進行討論，分別以顏色及辭例的完整度作為分類標準。以顏色劃分塗朱甲骨可分為三類：僅見塗朱者、朱墨褐三色同版者、朱墨或朱褐同版者。以辭例完整度可分成八類。 / The incised characters on oracle bones rubbed with cinnabar or ink are called cinnabar inscriptions or ink inscription. The vermillion colored cinnabar used on the coloring of oracle bones is said to have religious messages or aesthetic purposes. Previously limited by the immature color printing and computer technology, oracle bones inscriptions were mostly rubbing editions in black and white, and thus there are few in-depth studies on the coloring of oracle bone inscriptions. But now a corpus of oracle bones is taking form, such as editions with color pictures and digitalization of oracle bones images by Institute of History and Philosophy, Academia Sinica. This paper aims to examine the cinnabar inscriptions on oracle bones, discuss their characteristics in different periods, and categorize them into three categories based on colors, and eight categories based on the intactness of the inscriptions.

In summary, the biomechanical stimulations can augment osteotendinous healing processes by facilitating better fibrocartilagious transitional zone regeneration as well as the restoration of proprioceptions, and the early application showed the more beneficial effects. However, further experimental and clinical studies are still needed to explore the optimal timing, intensity, frequency, and duration of the proposed postoperative biomechanical stimulation protocols. / LIPUS is a "non-contact" biomechanical stimulation, which can provide a direct mechanical stimulation through cavitation and acoustic microstreaming effects to improve tissue healing in a less-than-rigid biomechanical environment. So the mechanical stimulation induced from LIPUS could be applied immediately after surgery without worrying about the mechanical strain exceed the structural property at the osteotendinous healing interface in the early phase of repair. In this part of study, we also examined the effects of the regime of biomechanical stimulations applying immediately after repair on the osteotendinous healing interface. By using the same healing junction model, forty-two female New Zealand white rabbits were randomly divided into two groups; daily mechanical stimulation was applied immediately after surgery lasting up to post-operative 12 weeks on the healing interface in the treatment group. The regime of mechanical stimulations included by LIPUS was 20 minutes, 5 days per week for 4 weeks, followed by cyclic mechanical stimulation generated from quadriceps muscles induced by FES for 8 weeks. Results showed that early application of biomechanical stimulations on the osteotendinous healing interface were significantly better radiologically, histologically and biomechanically than that of not any or later application of the biomechanical stimulations during the osteotendinous healing processes when assessing at the same healing time point. In addition, the early application of biomechanical stimulations showed the better functional recovery in terms of the restoration of the proprioceptions, which an increased numbers of sensory nerve endings labeled by calcitonin gene-relate peptide (CGRP) was detected in the whole osteotendinous healing complex. / Sports or trauma injuries around osteotendinous junctions are common; treatments usually require surgical reattachment of the involved tendon to bone. Restoration of osteotendinous junction after repair is slow and difficult due to regenerating the intermitted fibrocartilage zone to connect two different characteristic tissues, tendon to bone. Although the factors influencing fibrocartilage zone regeneration and remodeling during osteotendinous repair are poorly understood, however, is believed that the mechanical environment plays an important role in such healing process. In present study, the effects of mechanical stimulation on osteotendinous healing process were examined, in the way of mechanical stimulations induced by biophysical stimulations, surface functional electric stimulation (FES) and low intensity pulsed ultrasound (LIPUS), applying on the patellar tendon to patellar bone healing interface in an established partial patellectomy model in rabbits. / The mechanotransductive stimulation linked to the transmission of forces across osteotendinous junction can be generated from its muscle contraction induced by FES. In the partial patellectomy model, thirty-five female New Zealand white rabbits were randomly divided into two groups with initial immobilization for 6 weeks, daily FES was applied to quadriceps muscles for 30 minutes, 5 days per week for 6 weeks in treatment group and compared with non-treatment control group at postoperative week 6, 12 and 18, radiologically, histologically and biomechanically. Results showed that FES-induced cyclic mechanical stimulation significantly increased new bone formation and its bone mineral density. An elevated expression of tenascin C and TGFbeta1; an increased proteoglycant stainability; mature fibrocartilage zone formation with better resumptions of biomechanical properties also observed on the osteotendinous healing interface, indicating that the post-operative programmed cyclic mechanical stimulation generated from its muscle contraction has beneficial effects on osteotendinous healing processes by facilitating the fibrocartilagious transitional zone regeneration. / by Wang Wen. / Advisers: Kai Ming Chan; Ling Qin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1550. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 159-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Establishment of animal model for studying treatment efficacy of low-intensity pulsed ultrasound stimulations for accelerating bone-tendon repair. Standard partial patellectomy was conducted in the 18-week old rabbits that were then divided into the LIPUS treatment and control groups. The animals were followed for 2, 4, 8, and 16 weeks for various tissue analyses. LIPUS was applied to the experimental animals from postoperative day 3 to 16 weeks. We demonstrated that the healing process of PPT junction was initiated through endochondral ossification. The results showed that the size and length of newly formed bone, and its bone mineral content (BMC), but not its bone mineral density (BMD) were correlated with the failure load, ultimate strength and energy at failure. Using radiographic, biomechanical, histomorphologic and biomechanical methods, it was found that LIPUS had significant accelerating effect on PPT junction repair. We validated our study hypothesis in that LIPUS enhances bone-tendon junction healing by stimulating angiogenesis, chondrogenesis and osteogenesis. / Establishment of in vitro model for mechanism study on effects of low-intensity pulsed ultrasound stimulations. An in vitro model of osteoblast-like cell line (SaOS-2 cells) was studied using cDNA microarray to explore the molecular mechanism mediated by LIPUS. This microarray analysis revealed a total of 165 genes that were regulated at 4 and 24 hours by LIPUS treatment in osteoblastic-like cells. These genes belonged to more than ten protein families based on their function and were involved in some signal transduction pathways. This study has validated the hypothesis that LIPUS can regulate a number of critical genes transient expressions in osteoblast cell line Saos-2. / Keywords. partial patellectomy model; bone-tendon junction repair; low intensity pulsed ultrasound stimulations (LIPUS); gene expression; complementary DNA microarray; rabbit. / This study explored the intact morphology, regular healing and the augmented healing under the effects of low intensity pulsed ultrasound stimulations (LIPUS) on the patella-patella tendon (PPT) junction in a rabbit partial patellectomy model. To probe its possible mechanism, the key genes involved in regulating osteogenesis mediated by LIPUS were identified using the state-of-the-art methods---complementary DNA microarray. / Lu Hongbin. / "June 2006." / Advisers: Ling Qin; Kwok Sui Leung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1548. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 259-288). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

目前，骨形成低下的骨代謝異常在臨床中面臨巨大挑戰。治療這些疾病的途徑之一可通過小干擾核酸沉默骨形成抑制的基因。隨著核酸干擾技術的快速發展，採用核酸干擾策略進行治療的很多問題已被解決。然而，小干擾核酸的安全和有效遞送仍然是核酸干擾治療進行臨床轉化的瓶頸。其主要問題在於促進骨形成治療所需的小干擾核酸劑量較大，其系統給藥後可能對其他非骨組織產生副作用。所以，亟需針對具有促進成骨潛力的小干擾核酸開發安全有效的遞送系統。本研究的目的就是針對具有促進成骨潛力的小干擾核酸開發特定的遞送系統，以便應用於核酸干擾治療中的促進骨形成。策略之一是利用靶向骨形成表面的遞送系統攜載小干擾核酸到富集于骨形成表面的成骨系細胞。策略之二是直接把小干擾核酸遞送到成骨細胞，使其具有高度的細胞選擇性。在該研究中，我們採用具有成骨潛能的酪蛋白激酶2相互作用蛋白1小干擾核酸作為模型小干擾核酸以考察基因沉默效率。 / 靶向骨形成表面的(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統：首先對多肽序列(天門冬氨酸-絲氨酸-絲氨酸)₆靶向骨形成表面的特性進行鑒定。進一步將(天門冬氨酸-絲氨酸-絲氨酸)₆作為靶向分子與以DOTAP為主要成分的陽離子脂質體進行連接製備（天門冬氨酸-絲氨酸-絲氨酸)6-脂質體遞送系統。採用凍幹/再水化方法對小干擾核酸進行包裹並對其粒徑，ζ電位，包封率以及穩定性進行考察。最後分別在體外和體內模型對該遞送系統遞送效果以及其攜載小干擾核酸的基因沉默效率進行評價。 / 實驗結果證實(天門冬氨酸-絲氨酸-絲氨酸)₆是一種在體內可以有效靶向骨形成表面的多肽。(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體的平均粒徑為140 nm左右，其包封率可高達80%。該遞送系統較穩定，可使攜載的小干擾核酸具有較高的基因沉默效率，而且沒有明顯的細胞毒性。體內試驗表明，該遞送系統在促進小干擾核酸在骨組織的分佈同時降低其被肝組織的攝取。該遞送系統所攜帶的酪蛋白激酶2相互作用蛋白1小干擾核酸可選擇性地沉默骨組織中的酪蛋白激酶2相互作用蛋白1基因，且對其他組織並沒有明顯影響。該結果表明(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可促進小干擾核酸靶向骨組織並在骨組織沉默攜載小干擾核酸相應的基因。免疫化學分析結果顯示(天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體可攜載小干擾核酸選擇性地到達骨形成表面的成骨系細胞，避免被前破骨細胞/破骨細胞吞噬。大鼠骨髓細胞採用Alp，Stro-1和Oscar抗體分選後的酪蛋白激酶2相互作用蛋白1 mRNA表達水平顯示該遞送系統可選擇性地沉默成骨系細胞。 / 靶向成骨細胞的L6適配子-脂質納米顆粒-小干擾核酸遞送系統：將針對大鼠成骨細胞（ROS 17/2.8細胞系）進行正向篩選，大鼠肝細胞（BRL-3A細胞系）和外周血細胞進行負向篩選的L6適配子與以DLin-KC2-DMA為主要成分的脂質納米顆粒採用膠束形式插入的方法進行連接製備L6適配子-脂質納米顆粒-小干擾核酸遞送系統。並對其粒徑，ζ電位，包封率和形態學進行考察。在體外評價實驗中，考察了該遞送系統的選擇性，細胞毒性，基因沉默效率以及細胞攝取機制。在體內實驗中，對小干擾核酸的組織分佈以及其攜載小干擾核酸在成骨細胞和肝細胞的分佈進行了評價。 / 實驗結果顯示L6適配子-脂質納米顆粒-小干擾核酸的平均粒徑為84.0±5.3 nm，其電勢為-23 ± 2 mV，包封率為80.8 ± 3.4%. 脂質納米顆粒表面的L6適配子可促進小干擾核酸在ROS 17/2.8細胞系（靶向細胞）中的攝取， 然而在BRL-3A 細胞系（非靶向細胞）中攝入很少。該遞送系統沒有明顯細胞毒性，在10 nM小干擾核酸的低濃度下，體外基因沉默效率可高達50 % 以上。由L6適配子引起的巨胞被證實是成骨細胞攝取L6適配子-脂質納米顆粒所攜載小干擾核酸的主要機制。體內實驗顯示該遞送系統可促進小干擾核酸在骨組織的分佈，降低其被肝組織的攝取。在肝组织冰凍切片中，肝血竇和肝細胞中沒有明顯的小干擾核酸分佈，進一步說明該遞送系統可降低對肝組織的影響。免疫化學分析結果顯示L6適配子-脂質納米顆粒-小干擾核酸可攜載小干擾核酸選擇性地到達成骨細胞，避免被前破骨細胞/破骨細胞吞噬。 / 重要意義：本研究中的兩種新型小干擾核酸系統可分別選擇性地遞送小干擾核酸靶向骨形成表面和成骨細胞。 (天門冬氨酸-絲氨酸-絲氨酸)₆-脂質體-小干擾核酸遞送系統開拓了全新的途徑，實現選擇性地遞送小干擾核酸到骨形成表面從而降低對骨吸收的影響。 L6適配子-脂質納米顆粒-小干擾核酸遞送系統在成骨細胞表面特徵蛋白未知的情況下，首次採用適配子技術在細胞水準實現成骨細胞的選擇性遞送。該研究中的兩種遞送系統為核酸干擾治療的促進骨形成策略提供了強而有力的工具，為實現肌肉骨骼疾病相關領域的核酸干擾治療策略從基礎科學向臨床應用的轉化建立了堅實的基礎。 / Metabolic skeletal disorders that are associated with impaired bone formation are a major clinical challenge. One approach to treat these diseases was to silence bone formation-inhibitory genes by small interference RNAs (siRNAs). With the rapid development of RNA interference (RNAi) technology, more issues of RNAi-based therapy strategies have been addressed. However, the safe and effective delivery of siRNAs is still the bottleneck for its translation from bench to bedside. One major concern was that the large therapeutic doses of systemically administered siRNA to stimulate sufficient bone formation may carry a high risk for adverse effects on non-skeletal tissues. Therefore, development of specific siRNA delivery systems for safe and efficient transporting osteogenic siRNAs is highly desirable. The objective of the present study was to explore siRNA delivery systems for osteogenic siRNAs in RNAi-based bone anabolic therapy. One strategy was to develop siRNA delivery system targeting bone formation surfaces to facilitate delivery of siRNAs to osteogenic cells. Another approch was to develop siRNA delivery system targeting osteoblasts directly. Plekho1 siRNA targeting casein kinase-2 interacting protein-1 (Ckip-1) with osteogenic potential was employed as a representative siRNA in our current study. / (AspSerSer)6-liposome-siRNA for targeting bone formation surfaces: (AspSerSer)6 for targeting bone formation surfaces was firstly identified. Then, (AspSerSer)6 was conjugated with DOTAP-based liposome to produce (AspSerSer)6-liposome. (AspSerSer)6-liposome-siNRA was prepared by lyophilization/rehydration method and characterized in terms of particle size, zeta potential, encapsulation efficiency and the stability in serum. Finally, the delivery of siRNA and the corresponding gene silencing mediated by (AspSerSer)6-liposome-siRNA were evaluated in the in vitro and in vivo models. / The results indicated that the novel (AspSerSer)₆ was a promising peptide for targeting bone formation surfaces in vivo. (AspSerSer)₆-liposome with the average particle size of 140 nm encapsulating Plekho1 siRNA exhibited more than 80% encapsulation efficiency and good stability against enzymatic degradation. It demonstrated high knockdown efficiency without obvious cytotoxicity. In in vivo study, the result of tissue distribution experiment indicated that (AspSerSer)6-liposome-siRNA enhanced the distribution of siRNA in bone, meanwhile reduced the uptake of siRNA in liver. The Plekho1 protein and mRNA expression in various tissues demonstrated that (AspSerSer)₆-liposome-siRNA could facilitate gene silencing in a bone-selective manner. The results of immunochemistry analyses indicated (AspSerSer)₆-liposome-siRNA facilitated delivering siRNA to osteogenic cells at bone formation surfaces and avoided siRNA to pre-osteoclast/osteoclast. Plekho1 mRNA expression in rat bone marrow cells sorted by fluorescence activated cell sorting (FACS) using Alp, Stro-1 and Oscar antibody, respectively, further suggested (AspSerSer)₆-liposome-siRNA could silence gene in a cell-selective manner in vivo. / L6-LNPs-siRNA for targeting osteoblasts: L6 aptamer for targeting osteoblasts (ROS 17/2.8 cell line) and using rat hepatocyte (BRL-3A cell line) and peripheral blood cells in negative selection was conjugated to DLin-KC2-DMA-based lipid nanoparticles (LNPs) to generate L6-LNPs-siRNA by post-insertion method in the form of micelles. L6-LNPs-siRNA was characterized with particle size, zeta potential, encapsulation efficiency and morphology. Its selectivity, cytotoxicity and knockdown efficiency were evaluated in vitro. The mechanism of L6-LNPs-mediated siRNA cellular uptake was further investigated. The tissue distribution of the injected siRNA and the localization of the siRNA with osteoblasts as well as hepatocytes were also evaluated in vivo. / The results showed L6-LNPs-siRNA have the average particle size of 84.0 ± 5.3 nm and zeta potential of -23 ± 2 mV. Its encapsulation efficiency was 80.8 ± 3.4%. The L6 aptamer on the surface of LNPs facilitated the cellular uptake of Plekho1 siRNA in ROS 17/2.8 cell line (target cells) but no uptake in BRL-3A cell line (non-target cells) in vitro. L6-LNPs-siRNA with low cytotoxicity exhibited above 50% knockdown efficiency at a low concentration of 10 nM in vitro. Macropinocytosis induced by L6 was demonstrated to be the predominant mechanism of L6-LNPs mediated siRNA uptake in osteoblasts. In in vivo study, it was shown that L6-LNPs-siRNA facilitated the distribution of siRNA in bone and decreased the hepatic uptake. No obvious siRNA fluorescent signals in sinus and hepatocyte was observed in liver cryosection further indicated the reducing influence on liver after administration of L6-LNPs-siRNA. Co-localization of fluorescence-labeled siRNA with Alp-positive cells was dominantly documented, whereas there were no instances of such overlapping staining with Oscar-positive cells after L6-LNPs-siRNA treatment, which suggested L6-LNPs-siRNA facilitated delivering siRNA in a cell-selective manner in vivo. / Significance: These two innovative siRNA delivery systems in the present study selectively targeted bone formation surfaces and osteoblasts, respectively. (AspSerSer)₆-liposome-siRNA opened up a new avenue to specifically deliver therapeutic siRNAs to bone formation surfaces without affecting bone resorption. L6-LNPs-siRNA achieved the osteoblast-specific delivery for siRNA at cellular level by aptamer technology for the first time, even without knowledge of characteristic protein on the surface of osteoblasts. The two delivery systems provided the powerful tools for RNAi-based bone anabolic strategy and established a solid foundation for translating RNAi-based therapies from basic science to clinic applications in the musculoskeletal field. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wu, Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 130-142). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 論文摘要 --- p.vi / Table of contents --- p.ix / Publications --- p.xiv / List of tables --- p.xvi / List of figures --- p.xvii / List of abbreviations --- p.xxi / Chapter One Introduction --- p.1 / Chapter 1.1 --- Great challenges in skeletal disorders --- p.2 / Chapter 1.2 --- RNA interference (RNAi) as therapeutic strategy --- p.3 / Chapter 1.2.1 --- Mechanism of RNAi --- p.3 / Chapter 1.2.2 --- Potential triggers of RNAi-mediated gene silencing --- p.4 / Chapter 1.2.3 --- Current clinical trials using RNAi as therapeutic strategy --- p.7 / Chapter 1.2.4 --- Current application of therapeutic siRNAs in skeletal disorders --- p.11 / Chapter 1.3 --- Challenges of siRNA in vivo delivery for targeting bone --- p.12 / Chapter 1.3.1 --- General challenges of siRNA delivery in vivo --- p.13 / Chapter 1.3.2 --- Challenges of siRNA delivery to bone --- p.15 / Chapter 1.3.2.1 --- Physiological property --- p.15 / Chapter 1.3.2.2 --- Targeting ligands for approaching bone --- p.16 / Chapter 1.4 --- Strategies of siRNAs in vivo delivery after systemic administration --- p.18 / Chapter 1.4.1 --- Naked siRNA and naked siRNA with chemical conjugation --- p.18 / Chapter 1.4.2 --- Nanoparticle delivery systems --- p.20 / Chapter 1.4.2.1 --- Liposome and lipid-like materials --- p.20 / Chapter 1.4.2.2 --- Polymers --- p.22 / Chapter 1.4.2.3 --- Targeted delivery system --- p.23 / Chapter 1.5 --- Strategies of osteogenic siRNAs delivery for stimulating bone formation --- p.24 / Chapter 1.6 --- Objective of present study --- p.25 / Chapter Chapter Two --- Preparation and characterization of (AspSerSer)₆-liposome-siRNA for targeting bone formation surfaces --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Identification of (AspSerSer)₆ --- p.29 / Chapter 2.2.3 --- Development of formulation --- p.30 / Chapter 2.2.3.1 --- Selection of the molar ratio of DOTAP --- p.30 / Chapter 2.2.3.2 --- Selection of the molar ratio of siRNA to lipids --- p.30 / Chapter 2.2.4 --- Preparation of (AspSerSer)6-liposome-siRNA --- p.30 / Chapter 2.2.5 --- Characterization of (AspSerSer)₆-liposome --- p.33 / Chapter 2.2.5.1 --- Particle Size and Zeta Potential --- p.33 / Chapter 2.2.5.2 --- Encapsulation Efficiency --- p.33 / Chapter 2.2.5.3 --- Stability in serum --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- (AspSerSer)₆ as a targeting moiety --- p.34 / Chapter 2.3.2 --- Development of formulation --- p.37 / Chapter 2.3.3 --- Particle size, Zeta Potential and Encapsulation Efficiency --- p.38 / Chapter 2.3.4 --- Stability in serum --- p.38 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.5 --- Conclusion --- p.42 / Chapter Chapter Three --- Evaluation of (AspSerSer)₆-liposome-siRNA for cell-specific delivery and gene silencing in vitro and in vivo --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- Materials and Methods --- p.45 / Chapter 3.2.1 --- Materials --- p.45 / Chapter 3.2.2 --- Biological evaluation in vitro --- p.46 / Chapter 3.2.2.1 --- Binding affinity with hydroxyapatite --- p.46 / Chapter 3.2.2.2 --- Cell culture --- p.46 / Chapter 3.2.2.3 --- Cellular uptake --- p.47 / Chapter 3.2.2.4 --- Knockdown efficiency in vitro --- p.47 / Chapter 3.2.2.5 --- Total RNA extraction, reverse transcription and quantitative real-time PCR --- p.48 / Chapter 3.2.3 --- Cytotoxicity --- p.49 / Chapter 3.2.4 --- Tissue distribution --- p.50 / Chapter 3.2.4.1 --- Experimental design --- p.50 / Chapter 3.2.4.2 --- Fluorescence image analysis --- p.50 / Chapter 3.2.4.3 --- Quantitative Analysis --- p.50 / Chapter 3.2.5 --- Localization of siRNA in liver --- p.51 / Chapter 3.2.5.1 --- Experimental design --- p.51 / Chapter 3.2.5.2 --- Histochemisty analysis --- p.51 / Chapter 3.2.6 --- Gene silencing in tissues --- p.52 / Chapter 3.2.6.1 --- Experimental design --- p.52 / Chapter 3.2.6.2 --- Determination of mRNA expression --- p.52 / Chapter 3.2.6.3 --- Western blot analysis --- p.52 / Chapter 3.2.7 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.53 / Chapter 3.2.7.1 --- Experimental design --- p.53 / Chapter 3.2.7.2 --- Immunohistochemistry analysis --- p.53 / Chapter 3.2.8 --- Gene silencing at cellular levels --- p.54 / Chapter 3.2.8.1 --- Experimental design --- p.54 / Chapter 3.2.8.2 --- Flow cytometry cell sorting --- p.54 / Chapter 3.2.9 --- Statistical analysis --- p.55 / Chapter 3.3 --- Results --- p.56 / Chapter 3.3.1 --- Binding affinity with hydroxyapatite --- p.56 / Chapter 3.3.2 --- Cellular uptake --- p.57 / Chapter 3.3.3 --- Knockdown efficiency in vitro --- p.57 / Chapter 3.3.4 --- Cytotoxicity --- p.59 / Chapter 3.3.5 --- Tissue distribution by imaging analysis --- p.60 / Chapter 3.3.6 --- Quantitative analysis of tissue distribution --- p.62 / Chapter 3.3.7 --- Localization of siRNA in liver --- p.63 / Chapter 3.3.8 --- Plekho1 mRNA and protein expressions --- p.64 / Chapter 3.3.9 --- Immunohistochemistry analysis --- p.65 / Chapter 3.3.10 --- Gene silencing at cellular level --- p.71 / Chapter 3.4 --- Discussion --- p.74 / Chapter 3.5 --- Conclusion --- p.77 / Chapter Chapter Four --- Preparation and characterization of aptamer-functionalized lipid nanoparticle for siRNA cell-specific delivery --- p.78 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.80 / Chapter 4.2.1 --- Materials --- p.80 / Chapter 4.2.2 --- Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-di- oxolane (DLin-KC2-DMA) --- p.80 / Chapter 4.2.2.1 --- Synthesis of Linoleyl alcohol (1) --- p.81 / Chapter 4.2.2.2 --- Synthesis of Linoleyl bromide (2) --- p.81 / Chapter 4.2.2.3 --- Synthesis of Dilinoleylmethyl formate (3) --- p.82 / Chapter 4.2.2.4 --- Synthesis of Dilinoleyl Methanol (4) --- p.82 / Chapter 4.2.2.5 --- Synthesis of Dilinoleyl Ketone (5) --- p.83 / Chapter 4.2.2.6 --- Synthesis of 2, 2- Dilinoleyl- 4- (2-hydroxyethyl)-[1,3]-dioxolane (6) --- p.83 / Chapter 4.2.2.7 --- Synthesis of DLin-KC2-DMA --- p.83 / Chapter 4.2.3 --- Development of formulation --- p.84 / Chapter 4.2.3.1 --- Selection of the molar ratio of lipids --- p.84 / Chapter 4.2.3.2 --- Selection of the mass ratios of siRNA to lipids --- p.85 / Chapter 4.2.3.3 --- Selection of the molar ratios of L6-PEG2000-DSPE on L6-LNPs-siRNA --- p.85 / Chapter 4.2.4 --- Binding affinity with osteoblasts --- p.86 / Chapter 4.2.5 --- Preparation of L6-LNPs-siRNA --- p.86 / Chapter 4.2.5.1 --- Synthesis of L6-PEG2000-DSPE --- p.87 / Chapter 4.2.5.2 --- Preparation of LNPs-siRNA --- p.87 / Chapter 4.2.5.3 --- Post-insertion of aptamers on the surface of LNPs-siRNA --- p.88 / Chapter 4.2.6 --- Characterization of L6-LNPs-siRNA --- p.88 / Chapter 4.2.6.1 --- Particle size and Zeta Potential --- p.88 / Chapter 4.2.6.2 --- Encapsulation Efficiency (EE) --- p.88 / Chapter 4.2.6.3 --- Cryo-Transmission electron microscope --- p.89 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Synthesis of DLin-KC2-DMA --- p.90 / Chapter 4.3.2 --- Formulation development --- p.93 / Chapter 4.3.3 --- Preparation of L6-LNPs --- p.95 / Chapter 4.3.4 --- Characterization of L6-LNPs-siRNA --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.5 --- Conclusion --- p.101 / Chapter Chapter Five --- Evaluation of L6 aptamer functionalized lipid nanoparticles (L6-LNPs-siRNA) for osteoblast-specific delivery in vitro and in vivo --- p.102 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Materials and Methods --- p.103 / Chapter 5.2.1 --- Materials --- p.103 / Chapter 5.2.2 --- Biological evaluation in vitro --- p.104 / Chapter 5.2.2.1 --- Cell culture --- p.104 / Chapter 5.2.2.2 --- Binding affinity with target/non-target cells --- p.105 / Chapter 5.2.2.3 --- Cellular uptake of siRNA in target/non-target cells --- p.105 / Chapter 5.2.2.4 --- Knockdown efficiency in vitro --- p.105 / Chapter 5.2.3 --- Cytotoxicity --- p.106 / Chapter 5.2.4 --- Mechanism of cellular uptake --- p.106 / Chapter 5.2.4.1 --- Spectral bio-imaging for endocytic pathways --- p.106 / Chapter 5.2.4.2 --- Chemical inhibition for endocytic pathways --- p.107 / Chapter 5.2.4.3 --- Determination of membrane ruffling --- p.107 / Chapter 5.2.5 --- Evaluation of specific delivery in vivo --- p.107 / Chapter 5.2.5.1 --- Experimental design --- p.107 / Chapter 5.2.5.2 --- Tissue distribution --- p.108 / Chapter 5.2.5.3 --- Localization of siRNA in liver --- p.108 / Chapter 5.2.5.4 --- Localization of siRNA with osteoblast/osteoclast --- p.108 / Chapter 5.2.6 --- Statistical analysis --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Binding selectivity of L6-LNPs-siRNA --- p.109 / Chapter 5.3.2 --- Selectivity of siRNA cellular uptake --- p.111 / Chapter 5.3.3 --- Knockdown efficiency in vitro --- p.112 / Chapter 5.3.4 --- Cytotoxicity --- p.113 / Chapter 5.3.5 --- Mechanism of cellular uptake --- p.113 / Chapter 5.3.6 --- Tissue distribution --- p.118 / Chapter 5.3.7 --- Localization of siRNA in liver --- p.119 / Chapter 5.3.8 --- Localization of siRNA with Osteoblasts/Osteoclasts --- p.120 / Chapter 5.4 --- Discussion --- p.123 / Chapter 5.5 --- Conclusion --- p.125 / Chapter Chapter Six --- Summary of the study and future research --- p.126 / Chapter 6.1 --- Summary of the study --- p.127 / Chapter 6.2 --- Future research --- p.128 / References --- p.130

Osteoporosis is a skeletal disease affecting 44 million Americans. A primary strategy to prevent osteoporosis is to develop a high peak bone mass in youth. Oral Contraceptives (OCs) alter hormones in women and could affect bone mass development. The interaction between OCs and skeletal mineralization is poorly understood. PURPOSE: Our aims were to 1) compare bone mineral density (BMD) of young women who had a history of OC use with regularly menstruating controls, 2) compare changes in BMD in controls, women who initiate OC use, and those who have a history of use, and 3) to evaluate predictive capabilities of physical activity and years of oral contraceptives use on changes in BMD. METHODS: We recruited women, 18 to 25 years of age, with a history of OC use and controls. BMD at the hip, whole-body, and spine (AP, g/cm�� and width-adjusted lateral, g/cm��) was measured by dual-energy x-ray absorptiometry. Physical activity (METs) was measured via questionnaire and grip strength was evaluated using an isometric dynamometer. RESULTS: Groups were similar in body mass index (BMI), fat mass, grip strength, calcium intake and physical activity but controls were slightly older than OC users. In analysis of covariance (ANCOVA), controlling for age and BMI, controls had significantly greater BMD than OC users at baseline at the AP and lateral spine, hip, and whole-body (p<0.05). By ANCOVA (covariates = age at baseline, change in weight), oral contraceptive users had greater bone loss at L��� in the lateral view than controls whereas, controls had greater increases in L��� volumetric BMD, BMD of the total hip, and whole body than OC users (p<0.05). Stepwise regression results did not reveal years of oral contraceptive use, grip strength, or METs to be a significant predictor of changes in BMD at any site. CONCLUSIONS: We conclude that, in the cross-sectional analysis, oral contraceptive use by young women may compromise bone health during a time when mineral is still accruing. In the prospective analysis, regularly menstruating controls had greater BMD accrual or less bone loss over a 12-month time period than women with a history of oral contraceptive use. / Graduation date: 2005

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

Bone discolouration of pig carcasses is a quality concern that has been observed in Ontario slaughter plants. The objectives of this study were to establish the prevalence of pig carcasses showing bone discolouration, its relationship with residues of tetracyclines in bones, and to investigate the use of tetracyclines in feeding programs for grower-finisher pigs as the main risk factor for discolouration. Abattoir data were examined to determine the extent of the problem and the prevalence of bone discolouration during 2006, 2008, 2009, and 2010 was found to be 0.13%, 0.22%, 0.26%, and 0.28%, respectively, indicating that the issue of bone discolouration was present at low levels over the entire period of the study. A controlled trial using feed, water, and injectable tetracycline products to investigate the effect of tetracyclines on residue and bone colour was conducted. Bones were assessed visually for signs of discolouration, and high performance liquid chromatography (HPLC) was used to measure the levels of tetracycline residues in the bones. Results from this trial demonstrated that discolouration could be produced with 660ppm of chlortetracycline (CTC) in feed for 12 weeks even when 33 days of withdrawal time was observed. It was also found that residues of tetracyclines can be present in bones in the absence of discolouration. A retrospective study was conducted to investigate tetracycline use in herds identified as having discoloured bones at slaughter. Positive shipments were associated with dosage and duration of CTC use as well as with length of withdrawal. In conclusion, discoloured bones of pig carcasses were identified at low levels in one large Ontario abattoir; however, further investigation is needed in order to determine the impact it may have on the swine industry. / Ontario Pork and the University of Guelph - Ontario Ministry of Agriculture (OMAFRA) Sustainable Production System Program

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.