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There are at least two models describing how the enzymes of metabolic pathways are arranged in living cells. The first is a stochastic model, where enzymes are freely-diffusing in the aqueous environment of the cell, and the second, the metabolon model, has pathway enzymes organized as enzyme complexes. Both are valid scientific hypotheses in that they make predictions that can be tested regarding pathway regulation, localization, and function. The goal of the work presented here was to test the metabolon model using the flavonoid biosynthetic pathway in Arabidopsis, which has been hypothesized to exist as a metabolic enzyme complex. Five novel mutants of the gene encoding the first enzyme of flavonoid biosynthesis, chalcone synthase (CHS), were characterized in an effort to develop tools for investigating the organization of flavonoid metabolism in Arabidopsis. A variety of mutant CHS genotypes were identified in this allelic series, including ones that displayed both null and temperature-sensitive phenotypes, based on endproduct analysis. Characterization of protein and RNA levels indicated that the stability of the CHS enzyme was reduced in some of the mutants as compared to wild type. In several of the alleles, homodimerization of CHS was also impaired. Effects of the mutations at the amino acid level were predicted from the three-dimensional crystal structure of the highly-homologous alfalfa CHS, which indicated substitutions at diverse sites on the enzyme, including ones that may disrupt folding and/or active site function. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization. In an effort to determine the in planta location of the first two enzymes in flavonoid biosynthesis, CHS and chalcone isomerase (CHI), immunolocalization experiments were performed. Results indicate that CHS and CHI are abundant in epidermal and cortex cells of the root elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. At the subcellular level, both of these enzymes were found to localize to the endoplasmic reticulum (ER), consistent with the hypothesis that the enzymes of flavonoid biosynthesis are organized as a membrane-associated enzyme complex. Analysis of the tt7(88) mutant, which lacks the cytosolic domain of the putative 'anchor' P450 enzyme, flavonoid 3'-hydroxylase, showed an altered distribution of CHS and CHI as compared to wild type, however CHS and CHI were still found to be associated with ER. These results suggest that complex interactions occur within the flavonoid enzyme complex to mediate the subcellular distribution of its constituents. Also evident from these studies was the asymmetric distribution of CHS and CHI in cortex cells of the elongation zone, a finding that may provide clues about the physiological function of flavonoids in roots. Together, these immunolocalization data support the metabolon model for the organization of flavonoid biosynthesis in Arabidopsis. In an effort to develop tools to investigate the in vivo dynamics of flavonoid biosynthesis, fusion proteins between CHS or CHI and the reporter, green fluorescent protein (GFP), were produced. Transient transfection assays in epidermal cells from onion root bulbs and Arabidopsis seedlings indicated that the GFP component of the fusion constructs was functional, as determined via GFP fluorescence. To investigate the spatial and temporal dynamics of these fusion proteins in all cell types, Arabidopsis plants stably transformed with the CHI-GFP fusion constructs were generated. The analysis of these transgenic plants should provide information regarding the localization and dynamics of flavonoid biosynthesis in vivo, and thereby serve to offer new insights into the function and regulation of this important plant metabolic pathway. Overall, the research presented here represents a significant contribution toward understanding how subcellular organization may be important in regulating metabolism. / Ph. D.

Flavonoid biosynthesis is an important secondary metabolic pathway in higher plants with a range of vital functions in plants and animals. This pathway has been developed as a model system for the study of multi-enzyme complexes. The goal of the work presented here was to structurally characterize a series of loss-of-function chalcone synthase (CHS) alleles and to define the molecular basis of the interaction between CHS and the second enzyme of flavonoid biosynthesis, chalcone isomerase (CHI). CHS proteins encoded by five previously characterized alleles were characterized by homology modeling in an effort to explain the alterations in function, stability, and dimerization exhibited by these variants. Four of the encoded proteins have a single amino acid substitution and the fifth is a truncated protein resulting from a frameshift. Models for each of these proteins were generated in silico and analyzed after molecular dynamics simulations. This analysis suggested reasons for changes in catalytic ability and stability for three of the five CHS variants. To characterize the molecular basis of the CHS-CHI interaction, a model was developed using X-ray crystallography, small-angle neutron scattering (SANS), in silico docking, molecular dynamics simulations, and yeast 2-hybrid analyses. These enzymes appear to be interacting in a manner that could facilitate the flow of intermediates from one active site to another. These experiments also identified a series of amino acids that appear to be involved in the interaction, which are currently undergoing alteration and analysis using a yeast 2-hybrid assay to verify the authenticity of the model. The data presented herein could be used in future engineering experiments to alter pathway flux to control the levels or types of flavonoid endproducts, resulting in more nutritious plants or flowers with novel pigments. These experiments advance the study of the structure of multi-enzyme complexes, an area that currently contains little information. As well, this is the first known use of SANS for the investigation of the architecture of metabolons. The techniques described herein could easily be applied to other systems in an effort to better understand the organization of multi-enzyme complexes and the implications of these assemblies on metabolic regulation. / Ph. D.

No / Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Sustained activation of nuclear transcription factor κB (NF-κB) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-κB signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNFα, 150 ng/ml) increased NF-κB-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative IκBα construct. In addition, TNFα increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-κB activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((−)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1–1 μM) for 18 h. None of the flavonoids modulated constitutive or TNFα-induced NF-κB activity. Therefore, we conclude that NF-κB signalling in astrocytes is not a major target for flavonoids.

Aim. Baicalein is a bioactive flavonoid component isolated from the root of Scutellaria baicalensis, which has been used as a traditional Chinese medicinal herb for the treatment of inflammation for centuries. Although various pharmacological effects of baicalein have been demonstrated, only limited studies in rats reported pharmacokinetic of baicalein, which exhibited a low oral bioavailability due to extensive first-pass metabolism. In addition, no investigation on human oral absorption or metabolic kinetic profile was reported previously. The current project conducted a series of mechanistic studies aiming to elucidate the intestinal absorption, metabolism and disposition of baicalein. Since glucuronidation plays an important role in the first-pass metabolism of flavonoids including baicalein, additional studies on the relationship between human intestinal glucuronidation activities and chemical structures of flavonoids have also been performed. / Conclusion. Baicalein is well absorbed at intestine but subjected to extensive intestinal glucuronidation resulting in low oral bioavailability. The glucuronidation of baicalein is catalyzed by multiple UGT isozymes. The disposition of baicalein 7-O-glucuronide, the major metabolite of baicalein in vivo, is mediated by the MRP and OATP transporters. The nucleophilicity and stereo-conformation of -OH substituents are crucial for the intestinal glucuronidation of flavonoids. / Methods. For investigation on intestinal absorption, metabolism and disposition of baicalein, human Caco-2 cell monolayer model, rat in situ intestinal perfusion model, and in vitro metabolism model were employed in the present study. For the further investigation on the position preference on glucuronidation of flavonoids at human intestine, the in vitro rates of glucuronidation among seven commercially available mono-hydroxyflavones, namely 3-, 5-, 6-, 7-, 2'-, 3'- and 4'-mono-hydroxyflavones were determined and compared. / Results. The satisfactory permeabilities of baicalein obtained from both Caco-2 cell model and rat intestinal perfusion model indicated its potential good absorption at gastrointestinal tract. Therefore, absorption should not be the rate-limiting factor causing the low oral bioavailability of baicalein. However, extensive glucuronidation occurred in the rat intestine perfusion model with over 90% of baicalein being metabolized after intestinal absorption. Consistent findings were also observed in the in vitro enzyme kinetic studies of baicalein. The biotransformation of baicalein to baicalein 7-O-glucuronide was extensive in human liver microsome, human jejunum microsome, rat liver microsome, and rat jejunum microsome with intrinsic clearances (Vmax/Km) of 618, 446, 436, 298 mul/min/mg, respectively, which are orders of magnitude greater than those of most of western drugs that share the same metabolic pathway. Further enzyme kinetic studies using human recombinant glucuronosyltransferases (UGT) isozymes showed that UGT 1A1, 1A3, 1A8, 1A9, 1A7 and 2B15 were involved in the glucuronidation of baicalein with different kinetic profiles. Mechanistic studies on the disposition of baicalein 7-O-glucuronide formed from a rapid glucuronidation of baicalein in intestine demonstrated that this intracellularly formed glucuronide of baicalein could be actively extruded to both the apical and basolateral sides (the so called efflux) in Caco-2 cell model as well as rat intestinal perfusion model. It was also found that the efflux of the baicalein 7-O-glucuronide followed saturable enzyme kinetics and was effectively inhibited by multi-drug resistance associated proteins (MRP) and organic anion transporters (OATP) inhibitors. Further study on the relationship between flavonoid structures and glucuronidation activities using seven monohydroxyflavones demonstrated that the conjugation rates of 6- and 3'-monohydroxyflavones (HF) were much greater than those of 3-, 4'-, 7-, 2'-HF, while 5HF was the lowest. / Zhang Li. / "August 2006." / Advisers: Zhong Joan Zuo; Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1587. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 186-223). / 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.

Ampelopsis grossedentata is a wildly used herb in South China as sleep aid beverage for many years. Yet the active ingredients and mechanisms of this herb were unknown. In the present study, extract from Ampelopsis grossedentata which we named ECBRC-AG, and one of its active ingredient myricetin were proved having significant hypnotic/sedative effects in multiple animal models. ECBRC-AG shortened sleep latency, increase NREM sleep and decrease locomotor activity when treated before the onset of light period in rats. ECBRC-AG could decrease active awake and increase REM sleep in the late part of light period. ECBRC-AG also decreased the caffeine induced hyperactivity in rats. Among the three suspected active ingredients from ECBRC-AG, myricetin showed similar active profile with ECBRC-AG. Myricetin increased NREM and REM sleep, decreased sleep latency, decreased locomotor activity and also active awake. All the above evidences have implicated that myricetin is the most important active ingredient of ECBRC-AG ECBRC-AG and myricetin did not show any obvious side effects on rats. / Based on these findings, we propose that myricetin facilitates GABA function on PVN neurons through a T-type calcium channel and CaM-KII mechanism. The hypnotic/sedative effects of ECBRC-AG and myricetin are mediated by PVN. ECBRC-AG treatment decreased corticosterone levels in rats, which also indicated that PVN/HPA axis was the target of these herbal derivates. PVN has broad interactions with GABAergic, hypocretinergic, cytokine and NPY system and all these systems are proved to be deeply involved in sleep regulation. / In conclusion, the present study has identified that myricetin is the most important active ingredient of the herbal extract ECBRC-AG. We confirmed the hypnotic/sedative effects of ECBRC-AG and myricetin on rats, and also revealed the different action profiles of these herbal derivates compared with zolpidem. T-type calcium channels and the HPA axis were shown to be involved in the mechanisms of ECBRC-AG and myricetin, indicating that they may be the new targets for insomnia treatment with these herbal derivates. / Insomnia is the most common sleep disorder and affects about one third of the general population. Insomnia is always combined with physical and mental illness, as either a consequence or a contributing factor. Insomnia produces sleepiness, impairment in psychomotor performance, absenteeism, frequent accidents, memory impairment and a high risk of depression. Pharmacologic therapies are the most important interventions for insomnia. However, the currently available hypnotics are associated with residual effects and risks of abuse and dependence. More efficient and safe hypnotics are needed. / The DNA array and RT-PCR studies revealed that GABA, hypocretin, cytokine and NPY systems were involved in the mechanisms of ECBRC-AG and myricetin. In calcium imaging study, we found that myricetin induced a transient Ca 2+ influx in the primary culture of rat hypothalamus neurons. This Ca2+ influx could be blocked by T-type channel blocker mibefradil. RT-PCR study also showed that ECBRC-AG and myricetin treatment changed the mRNA expression level of T-type calcium channel al G subunit in rat hypothalamus. The present results are consistent with our previous study showing that myricetin enhanced GABA function in the neurons of rat hypothalamic paraventricular nucleus (PVN), and that blocking CaM-KII pathway eliminated this effect. / Zhang, Xiaohu. / "March 2008." / Adviser: Chan Hsiao Chang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1516. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 156-174). / 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.

Yip Wai Nga. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 93-101). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.v / Acknowledgements --- p.viii / Table of contents --- p.ix / List of figures --- p.xii / List of abbreviation --- p.xv / Chapter Chapter I: --- Introduction --- p.1 / Chapter I.1 --- Transepithelial ion transport --- p.1 / Chapter I.1.1 --- Fluid secretion in colon --- p.1 / Chapter I.1.2. --- Cellular mechanism of chloride secretion --- p.3 / Chapter 1.2. --- Regulation of chloride secretion in T84 cells --- p.6 / Chapter I.2.1 --- Human colonic T84 cells as the study model --- p.6 / Chapter I.2.2 --- Signal transduction pathways of chloride secretion in T84 cells --- p.13 / Chapter I.3. --- Pharmacological actions of Scutellariae Radix --- p.13 / Chapter I.3.1. --- What is Scutellariae Radix? --- p.13 / Chapter I.3.2. --- Some biological and pharmacological actions of Scutellariae Radix --- p.13 / Chapter I.4. --- Effects of Scutellariae Radix and its major flavonoid baicalein on ion transport in T84 cells --- p.15 / Chapter I.4.1. --- Effects of Scutellariae Radix extract on ion transport in T84 cells --- p.15 / Chapter I.4.2. --- Biological effects of baicalein --- p.15 / Chapter I.5 --- "Relationship of Coptidis rhizoma and its active ingredient berberine, with Scutellariae radix in traditional remedies" --- p.18 / Chapter I.6 --- Aim of study --- p.20 / Chapter Chapter II: --- Methods and Materials --- p.21 / Chapter II.1. --- Culture technique of the T84 cells --- p.21 / Chapter II.2. --- Conventional short-circuit current (Isc) measurement --- p.24 / Chapter II.2.1. --- Experimental setup --- p.24 / Chapter II.2.2. --- Preparation of the permeable supports --- p.27 / Chapter II.2.3. --- Cell seeding --- p.27 / Chapter II.2.4. --- Short-circuit measurement --- p.29 / Chapter II.2.5 --- Short-circuit measurement in nystatin-permeabilized T84 monolayers --- p.30 / Chapter II.3. --- Measurement of protein kinase A activity --- p.31 / Chapter II.4. --- Solutions and chemicals --- p.32 / Chapter II.5. --- Statistical analysis --- p.33 / Chapter Chapter III: --- Result --- p.34 / Chapter III.1. --- Effect of SRE on transepithelial ion transport processes in T84 monolayers --- p.35 / Chapter III.1.1 --- Effect of SRE and baicalein on baseline Isc --- p.35 / Chapter III.1.2 --- Effect of ion channel blockers on SRE-stimulated Isc --- p.39 / Chapter III.1.3 --- Effect of K+ channel blockers on SRE-stimulated Isc --- p.42 / Chapter III.1.4 --- Effect of SRE in C1- free solution --- p.48 / Chapter III.2. --- Effect of SRE on apical C1- conductance and basolateral K+ conductance in nystatin-permeabilized T84 monolayers --- p.51 / Chapter III.2.1 --- Effect of SRE and baicalein on baseline IC1 --- p.51 / Chapter III.2.2 --- Study of apical C1- conductance in T84 monolayers --- p.54 / Chapter III.2.3 --- Interaction of SRE and forskolin --- p.61 / Chapter III.2.4 --- Study of basolateral K+ conductance in T84 monolayers --- p.62 / Chapter III.3 --- Effect of SRE and baicalein on PKA activities in T84 cells --- p.64 / Chapter III.3.1 --- Effect of Scutellariae Radix on PKA activity --- p.66 / Chapter III.3.2 --- Effect of baicalein on PKA activity --- p.69 / Chapter III.3.3. --- Effect of berberine on PKA activity --- p.69 / Chapter III.3.3. --- Interaction of baicalein and berberine on PKA activity --- p.74 / Chapter Chapter IV: --- Discussion --- p.77 / Chapter IV.1 --- "Scutellariae Radix，Coptidis Rhizoma, and gastrointestinal secretory function" --- p.77 / Chapter IV.2 --- SRE- and baicalein-induced increase in Isc --- p.79 / Chapter IV.3 --- Cellular signaling mechanisms underlying the effect of SRE and baicalein --- p.82 / Chapter IV.4 --- "Interaction between Scutellariae Radix and Coptidis Rhizoma - the ""ying and yang"" hypothesis" --- p.89 / Chapter IV.5 --- Summary --- p.91

研究背景：常规骨科临床在治疗大段骨缺损时需要移植骨和（或）支架材料，尤其复合有治疗性生物活性成分的复合材料尤为理想。本研究的策略在于发展开发一种具有生物活性和生物降解特性的的合并有植物小分子icaritin（外源性生长因子）或者骨形态发生蛋白2（BMP-2, 内源性生长因子）的复合骨支架用于骨再生。基于聚乳酸乙交酯共聚物和磷酸三钙，我们利用先进的快速成型技术编制了新型的符合有BMP-2 或者icaritin 的支架材料， 命名为PLGA/TCP （ 对照材料组） ，PLGA/TCP/BMP-2（BMP-2 编织复合治疗材料组）， PLGA/TCP/icaritin （低，中，高剂量icaritin 编织复合治疗材料组）。 / 研究目标：本研究的总体目标是通过系统的体外实验和兔骨缺损的体内实验，建立和评估一种优化的复合递送系统，用于骨再生的应用。体内效果的研究体现在终点关于合并有外源性生长因子icaritin 和内源性生长因子BMP-2 的复合材料之间的比较研究。 / 材料和方法：低温快速成型机器用于复合材料的编制。PLGA 和TCP 作为基本载体材料，icaritin 和BMP-2 作为具有生物活性的外源性和内源性生长因子，分别进行编织复合。最终编织复合的支架材料命名为P/T 对照组，P/T/BMP-2 和低，中，高剂量P/T/icaritin 治疗组。另外，我们通过液体完全浸泡并在真空橱内干燥24 小时的方法制备了BMP-2 和icaritin 浸泡复合支架材料，分别是P/T+BMP-2(阳性对照组)和中剂量P/T+icaritin(比较组)。体外成骨潜能是通过兔骨髓干细胞和支架材料共培养的方法检测细胞接种，增殖效率，碱性磷酸酶活性，钙沉积以及成骨基因定量mRNA 表达检测。兔尺骨双侧阶段性缺损并植入复合支架材料的模型用于探讨支架材料体内成骨和成血管功效，影像学和活体检测CT 技术用于评估骨再生；借助CT的血管造影术和组织学检测新生血管；动态核磁共振技术用于检测骨缺损局部血液灌注功能，以及宿主组织和支架材料之间的相互作用。 / 研究结果： 对编织的支架材料的体外特性和成骨潜能进行鉴定和评估。显微CT 定量结果显示此支架材料具有互联大孔隙，平均孔隙率75±3.27%，平均孔径458±25.6μm。和对照组，icaritin 浸泡复合组，BMP-2 编织复合组比较，在icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组（n=6, p<0.01）中，与材料共培养的兔骨髓干细胞（BMSCs）表现了较高的细胞接种效率，碱性磷酸酶活性和上调的胶原酶I，骨桥蛋白mRNA 表达，以及较多的钙结节沉积。同时，BMP-2 浸泡复合组表现了最佳的效果（n=6, p<0.01）。兔尺骨缺损模型体内试验结果显示，术后2，4，8周影像学和显微CT 显示，和对照组，icaritin 浸泡复合组，BMP-2 编织复合组比较，icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组材料（n=6, p<0.01）植入的骨缺损区域有更多新生成骨。BMP-2 浸泡复合组表现了最多的新骨形成（n=6,p<0.01）。组织学结果同样也验证了在icaritin 编织复合支架材料（n=6, p<0.05）特别是中剂量组（n=6, p<0.01）中，存在较多的骨样组织和典型的板层骨。BMP-2 浸泡复合组也具有最多的新骨组织生成（n=6, p<0.01）。此外， 在icaritin 编织复合支架材料（n=6, p<0.05）尤其中剂量组（n=6, p<0.01）中，借助显微CT 的血管造影术检测发现，骨缺损区域出现较大的新生血管体积，动态核磁共振检查发现较好的局部血液灌注功能。在三种icaritin 剂量浓度的编织复合材料组之间比较，我们发现中浓度icaritin 复合比例的编织复合材料组显示了最佳的成骨潜能。 / 研究结论： 编织复合有外源性植物分子icaritin 的PLGA/TCP 支架材料在体内体外试验中均表现了预期的成骨分化潜能和骨再生能力，尤其是中剂量icaritin 编织复合材料。传统的应用前做体外复合的BMP-2 浸泡复合支架材料和更具吸引力和方便应用的植物分子icaritin 编织复合支架材料，都可以较好的增强骨修复，这很可能为新型生物复合材料潜在的临床有效性验证提供很好的基础。 / Background: Treatment of large bone defect in routine orthopaedic clinics requires bonegrafting and/or scaffold materials, especially desirable with composite material combined with therapeutic and bioactive agents for achieving better treatment outcome. The strategy of this study was to develop such a bioactive biodegradable composite bone scaffold incorporating a phytomolecule icaritin as an exogenous growth factor or bone morphogenetic protein-2 (BMP-2) as a known endogenous growth factor for bone regeneration. Based on polylactide-co-glycolide (PLGA) and Tricalcium Phosphate (TCP), we fabricated innovative BMP-2 or icaritin incorporated scaffold materials, namely PLGA/TCP (Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin with three different dosages of icaritin (Treatment groups) by an advanced prototyping technology. / Aims: The overall aim of the study was to establish and evaluate a local delivery system with slow release of bioactive agents for acceleration of bone regeneration in a bone defect model in rabbits. In vivo efficacy study served as end-point of this comparative study between composite scaffold incorporating exogenous growth factor icaritin and endogenous growth factor BMP-2. / Materials & Methods: Composite scaffolds were fabricated at -28ºC by a lowtemperature rapid-prototyping machine. PLGA and TCP were used as basic carrier materials, and icaritin or BMP-2 was incorporated as exogenous or endogenous bioactive growth factors, respectively. The incorporated scaffolds were named by PLGA/TCP (P/T, Control group), PLGA/TCP/BMP-2 and PLGA/TCP/low-, middle-, and high-icaritin (Treatment groups). In addition, we prepared BMP-2 and icaritin loading scaffolds, namely PLGA/TCP+BMP-2 as positive control group and PLGA/TCP+middle-icaritin as comparative group by entire immersion in the solution and dry in vacuum cabinet for 24 hours. In vitro osteogenic potentials of the designed bioactive composite scaffolds were tested in scaffold-co-cultured rabbit bone marrow stem cells (BMSCs) for measurement of cell seeding and proliferation efficiency, alkaline phosphatase (ALP) activity, calcium deposition, and quantitative mRNA expression of relative osteogenic genes. In vivo efficacy investigation was designed to evaluate osteogenesis and angiogenesis in a bilateral ulna bone segmental defect model implanted with composite scaffold in rabbits, with radiography and in vivo micro-CT for studying new bone regeneration and micro-CT-based angiography and histology for neovascularization, dynamic MRI for local blood perfusion function, as well as host tissue and scaffold material interactions. / Results: The in vitro characterization and osteogenic potential of the fabricated scaffolds were performed and confirmed, respectively. Micro-CT quantitation showed that the scaffolds had interconnected macropores with an average porosity of 75±3.27 % and pore size or diameter of 458±25.6 μm. Compared to P/T, P/T+icaritin and P/T/BMP-2 scaffolds, P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin (n=6, p<0.01) presented higher cell seeding efficiency, ALP activity and calcium nodules and up-regulated mRNA expressions of Collagen type I and Osteopontin of co-cultured BMSCs. P/T+BMP-2 showed the best osteogenic effects among all groups (n=6, p<0.01). In vivo measurement of x-ray and micro-CT in rabbit ulna bone defect model at week 2, 4 and 8 post-surgery showed more newly formed bone in the defects treated with P/T/icaritin scaffolds (n=6, p<0.05), especially P/T/middle-icaritin scaffold (n=6, p<0.01) compared with that of P/T, P/T+icaritin and P/T/BMP-2 groups. P/T+BMP-2 also showed the best bone formation among all groups (n=6, p<0.01). Histological results also demonstrated that there were more osteoid tissues and typical lamellar bone in surface and internal of the implants, as well as along the adjacent host bone in P/T/icaritin groups (n=5, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01). P/T+BMP-2 group showed the most newly formed bone (n=6, p<0.01). In addition, newly formed vessels in the defects were identified with micro-CT-based angiography and functionally supported by dynamic MRI for reflecting blood perfusion. The results showed more ingrowing new vessels in P/T/icaritin groups (n=6, p<0.05), especially P/T/middle-icaritin group (n=6, p<0.01), compared to P/T and P/T/BMP-2 groups. For comparing dose effects among three scaffolds incorporating different concentration of icaritin, we found that middle dose PLGA/TCP/icaritin composite scaffold showed the best osteogenic potential. / Conclusion: PLGA/TCP scaffolds incorporating exogenous phytomolecule icaritin demonstrated the desired osteogenic differentiation potential and bone regeneration capability as investigated in vitro and in vivo, where the middle dose of icaritin incorporating PLGA/TCP composite scaffold showed the best effects. These findings may form a good foundation for potential clinical validation of this innovative bioactive composite scaffold with either conventional endogenous BMP-2 for in vitro loading before application or more attractively and user-friendly incorporated with exogenous phytomolecule icaritin as a ready product for enhancing bone defect repair. / 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. / Chen, Shihui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 173-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.viii / Abstract --- p.x / 中文摘要 --- p.xiii / List of Abbreviations --- p.xvi / List of Tables --- p.xix / List of Figures --- p.xx / Journal Publications --- p.xxv / Journal Supplements --- p.xxv / Conference Abstracts --- p.xxvi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Bone Defect in Orthopaedics --- p.2 / Chapter 1.2 --- Human Skeletons --- p.2 / Chapter 1.2.1 --- Bone Types and Function --- p.2 / Chapter 1.2.2 --- Bone Development --- p.4 / Chapter 1.2.3 --- Bone Physiology and Structure --- p.6 / Chapter 1.2.4 --- Bone Specific Markers --- p.7 / Chapter 1.2.5 --- Bone Cells --- p.9 / Chapter 1.2.6 --- Bone Marrow Stromal Cells --- p.12 / Chapter 1.3 --- Bone Regeneration and Remodeling --- p.13 / Chapter 1.3.1 --- Bone Defect Healing --- p.13 / Chapter 1.3.2 --- Non-union and Segmental Defect --- p.15 / Chapter 1.3.3 --- Bone Defect Treatment --- p.16 / Chapter 1.4 --- Angiogenesis in Bone Healing --- p.19 / Chapter 1.4.1 --- Blood Vessels Formation Process --- p.20 / Chapter 1.4.2 --- Growth Factor in Angiogenesis --- p.21 / Chapter 1.5 --- Biomaterials in Bone Tissue Engineering --- p.22 / Chapter 1.6 --- Scaffold-Based Therapy --- p.23 / Chapter 1.6.1 --- Bone Grafts --- p.23 / Chapter 1.6.1.1 --- Autografts --- p.23 / Chapter 1.6.1.2 --- Allografts --- p.25 / Chapter 1.6.2 --- Bone Graft Substitutes --- p.25 / Chapter 1.6.2.1 --- Bone Formation in Porous Scaffolds --- p.25 / Chapter 1.6.2.2 --- Degradable Polymers --- p.27 / Chapter 1.6.2.3 --- Non-Degradable Polymers --- p.29 / Chapter 1.6.2.4 --- Ceramics --- p.29 / Chapter 1.6.2.5 --- Bioactive Composite Materials --- p.30 / Chapter 1.7 --- Growth Factor-Based Therapy --- p.31 / Chapter 1.7.1 --- Endogenous Growth Factor--Bone Morphogenetic Proteins --- p.31 / Chapter 1.7.2 --- Exogenous phytomoleculeIcaritin--Icaritin --- p.31 / Chapter 1.7.3 --- Delivery of Growth Factor in Tissue Engineering --- p.34 / Chapter 1.8 --- Fabrication of Porous Composite Scaffolds --- p.37 / Chapter 1.8.1 --- Architectural Parameters of Bone Scaffolds --- p.37 / Chapter 1.8.2 --- Three-Dimensional Scaffold Fabrication --- p.37 / Chapter 1.9 --- Animal Models for Testing Bone Defects Healing --- p.39 / Chapter Chapter 2 --- Research Rationale and Study Objectives / Chapter 2.1 --- Research Rationale --- p.42 / Chapter 2.2 --- Study Objectives --- p.46 / Chapter Chapter 3 --- Bioactive Composite Scaffolds: Preparation, Morphology and Release Assay / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.50 / Chapter 3.2.1 --- Materials --- p.50 / Chapter 3.2.2 --- Fabrication of PLGA/TCP Incorporating BMP-2 or Icaritin --- p.51 / Chapter 3.2.3 --- Morphological Analysis of Composite Scaffolds --- p.53 / Chapter 3.2.3.1 --- Analysis of Porosity and Macropores Diameter Using High-resolution Micro-CT --- p.53 / Chapter 3.2.3.2 --- Analysis of Surface Morphology and Elements Composition Using Scanning Electron Microscopy --- p.54 / Chapter 3.2.4 --- Icaritin Content Assay in PLGA/TCP Scaffolds Incorporating Icaritin --- p.54 / Chapter 3.2.5 --- Preparation of PLGA/TCP Scaffold Coating BMP-2 or Icaritin --- p.55 / Chapter 3.2.6 --- In vitro Release Assay --- p.55 / Chapter 3.2.6.1 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.55 / Chapter 3.2.6.2 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.56 / Chapter 3.2.7 --- Mechanical Properties of Composite Scaffolds --- p.56 / Chapter 3.2.8 --- Statistical Analysis --- p.57 / Chapter 3.3 --- Results --- p.57 / Chapter 3.3.1 --- Morphological Analysis of Composite Scaffolds --- p.57 / Chapter 3.3.1.1 --- Porosity and Macroscopic Diameter --- p.57 / Chapter 3.3.1.2 --- Surface Morphology and Elements Composition --- p.58 / Chapter 3.3.2 --- Icaritin Content in Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60 / Chapter 3.3.3 --- Icaritin Release from Scaffolds of PLGA/TCP Incorporating Icaritin --- p.60 / Chapter 3.3.4 --- BMP-2 Release from Scaffolds of PLGA/TCP Incorporating/Coating BMP-2 --- p.61 / Chapter 3.3.5 --- Mechanical Properties of Composite Scaffolds --- p.63 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.5 --- Summary --- p.71 / Chapter Chapter 4 --- Bioactive Composite Scaffolds: In vitro Degradation and Characterization Studies / Chapter 4.1 --- Introduction --- p.73 / Chapter 4.2 --- Materials and Methods --- p.74 / Chapter 4.2.1 --- Preparation of Composite Scaffolds for in vitro Degradation Assay --- p.74 / Chapter 4.2.2 --- Characterizations --- p.75 / Chapter 4.2.2.1 --- Scaffold Volume Changes --- p.75 / Chapter 4.2.2.2 --- Scaffold Weight Changes --- p.75 / Chapter 4.2.2.3 --- pH Value Changes --- p.75 / Chapter 4.2.2.4 --- Calcium Ion Release from Scaffolds --- p.76 / Chapter 4.2.3 --- Mechanical Properties Changes --- p.76 / Chapter 4.2.4 --- Statistical Analysis --- p.77 / Chapter 4.3 --- Results --- p.77 / Chapter 4.3.1 --- Volume Decrease --- p.78 / Chapter 4.3.2 --- Weight Loss --- p.78 / Chapter 4.3.3 --- pH Value Reduction --- p.79 / Chapter 4.3.4 --- Calcium Ion Release --- p.79 / Chapter 4.3.5 --- Mechanical Properties --- p.80 / Chapter 4.4 --- Discussion --- p.81 / Chapter 4.5 --- Summary --- p.84 / Chapter Chapter 5 --- In vitro Evaluation of Bone Marrow Stem Cells (BMSCs) Growing on Bioactive Composite Scaffolds / Chapter 5.1 --- Introduction --- p.87 / Chapter 5.2 --- Materials and Methods --- p.90 / Chapter 5.2.1 --- Preparation of Composite Scaffolds for in vitro Evaluation --- p.90 / Chapter 5.2.2 --- BMSCs Seeding Rate and Proliferation on Composite Scaffolds --- p.90 / Chapter 5.2.3 --- Alkaline Phosphate (ALP) Activity Assay --- p.92 / Chapter 5.2.4 --- Osteogenic Gene Expression Assay Using Quantitative Real-time PCR --- p.92 / Chapter 5.2.5 --- Calcium Deposition Assay Using Alizarin Red Staining --- p.93 / Chapter 5.2.6 --- Statistical Analysis --- p.94 / Chapter 5.3 --- Results --- p.94 / Chapter 5.3.1 --- Cells Seeding Efficiency and Proliferation --- p.94 / Chapter 5.3.2 --- ALP Activity --- p.97 / Chapter 5.3.3 --- Osteogenic Gene mRNA Expression --- p.97 / Chapter 5.3.4 --- Calcium Deposition --- p.98 / Chapter 5.4 --- Discussion --- p.99 / Chapter 5.5 --- Summary --- p.102 / Chapter Chapter 6 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds / Chapter 6.1 --- Introduction --- p.105 / Chapter 6.2 --- Materials and Methods --- p.106 / Chapter 6.2.1 --- Preparation of Composite Scaffolds for Implantation --- p.106 / Chapter 6.2.2 --- Establishment of Ulna Bone Segmental Defect in Rabbits --- p.107 / Chapter 6.2.3 --- Radiographic Evaluation of New Bone Area Fraction --- p.109 / Chapter 6.2.4 --- XtremeCT Evaluation of New Bone Formation and Bone Mineral Density (BMD) --- p.110 / Chapter 6.2.5 --- Histological Evaluation of New Bone Formation --- p.111 / Chapter 6.2.6 --- Evaluation of Rate of New Bone Formation and Mineral Apposition Rate (MAR) --- p.114 / Chapter 6.2.7 --- Evaluation of Neovascularization Using Micro-CT-based Microangiography --- p.116 / Chapter 6.2.8 --- Blood Perfusion Function Using Dynamic Magnetic Resonance Imaging (MRI) --- p.119 / Chapter 6.2.9 --- Statistical Analysis --- p.120 / Chapter 6.3 --- Results --- p.121 / Chapter 6.3.1 --- Radiographic Area Fraction of New Bone Formation --- p.123 / Chapter 6.3.2 --- XtremeCT New Bone Volume Fraction and BMD --- p.128 / Chapter 6.3.3 --- Histological New Bone Fraction --- p.133 / Chapter 6.3.4 --- Rate of New Bone Formation and MAR --- p.136 / Chapter 6.3.5 --- New Vessels Volume Evaluated Using Micro-CT-Based Microangiography --- p.140 / Chapter 6.3.6 --- Dynamic Blood Perfusion Function --- p.144 / Chapter 6.4 --- Discussion --- p.146 / Chapter 6.5 --- Summary --- p.151 / Chapter Chapter 7 --- Summaries, Conclusions, Limitations and Future Studies / Chapter 7.1 --- Introduction --- p.153 / Chapter 7.2 --- Bioactive Composite Scaffolds: Preparation, Morphology and in vitro Release Evaluation --- p.155 / Chapter 7.3 --- Bioactive Composite Scaffolds: in vitro Degradation and Characterization Studies --- p.159 / Chapter 7.4 --- In vitro Evaluation of the Response of Bone Marrow Stem Cells Growing on Bioactive Composite Scaffolds --- p.160 / Chapter 7.5 --- In vivo Evaluation of Bone Healing in Bone Defect Model Implanted with Bioactive Composite Scaffolds --- p.162 / Chapter 7.6 --- Evaluation of Dose-dependent Effects of Icaritin Mechanical Property, Degradation, and Osteogenic Potentials --- p.164 / Chapter 7.7 --- Conclusions --- p.170 / Chapter 7.8 --- Limitations and Future Studies --- p.171 / Chapter 7.9 --- References --- p.173 / Chapter 7.10 --- Appendix --- p.199 / Chapter 7.10.1 --- Animal Licence and Ethics --- p.199 / Chapter 7.10.2 --- Safety Approval --- p.201 / Chapter 7.10.3 --- Journal Supplements --- p.202 / Chapter 7.10.4 --- Conference Abstracts--Posters --- p.205 / Chapter 7.10.5 --- Conformation of Paper Submission --- p.208 / Chapter 7.10.6 --- Published Paper --- p.209

"December 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 221-233) / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Steroid-associated osteonecrosis (SAON) accounts for a major fraction in non-traumatic osteonecosis (ON) and generally has poor prognosis even after surgical interventions. This suggests that prevention is one of the important intervention strategies for SAON. So far, there is lacking of proven prevention modalities for SAON. / Study I was to establish an alternative SAON model. Based on the proposed pathogenesis of SAON that the intravascular thrombosis and extravascular marrow fat deposition are the two important contributors to the development of ON lesion, lipopolysaccharides (LPS) could induce vascular dysfunction and even thrombosis, and methylprednisolone (MPS) could induce the adipogenesis of marrow mesenchymal stem cells (MSCs). They were accordingly used in a combination for ON induction in animals. / Study II was to investigate the effect of Herbal Epimedium-derived formula for prevention of ON using the validated SAON animal model. Efficacy of the herbal Epimedium-derived formula was assessed for prevention of SAON using the animal model. Thirty adult male rabbits were used in this study. The ON incidence was set as the end-point for evaluation of the prevention efficay. For the potential intervention targets, the intravascular thrombosis and extravascular marrow fat formation were evaluated hematologically and histopathologically. The vascular structure and function were evaluated by advanced bioimaging modalities of micro-CT and MRI. / Study III was to investigate the bioactive compound(s) from the herbal Epimedium-derived herbal formula for prevention of SAON. Phytochemical analysis identified seven compounds in this efficacy-proven formula, with icariin as the major compound accounting for more than 80% in weight. Icariin was therefore tested for its prevention efficacy using the SAON animal model. / Study IV was to investigate the underlying mechanism(s) of bioactive compound Icariin in effective prevention of SAON using in vitro cell models. As activation of endothelial cells and adipogenesis of MSCs are suggested to be the two major events involving in vascular dysfunction and marrow fat formation in SAON animal model, Icariin were accordingly hypothesized to be able to prevent activation of endothelial cells and inhibit adipogenesis of MSCs. / Summary. After summarizing the major findings of these four logically interrelated studies, it was able to conclude that Icariin was the identified bioactive compound from the herbal Epimedium-derived formula, which was able to reduce the SAON incidence with inhibition of intravascular thrombosis and extravascular marrow fat formation in an established rabbit model. The underlying mechanisms might be related to its effects on protection of endothelial cells activation and inhibition of MSCs adipogenesis (This can be summarized in the following picture). This study provides a new bioactive agent Icariin for SAON prevention and potential future clinical application. (Abstract shortened by UMI.) / The following research questions were addressed in the present study: (1) Is there an alternative SAON animal model? (Study I); (2) Whether the herbal Epimedium-derived formula is able to prevent SAON in this animal model? (Study II); (3) What is the bioactive compound(s) in this herbal Epimedium-derived formula? (Study III); (4) How does this bioactive compound prevent SAON? (Study IV) / Sheng, Hui. / Adviser: Ling Qin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3421. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / 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.