by Yurianto Henry. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 88-93). / ABSTRACT --- p.I / ACKNOWLADGEMENT --- p.IV / TABLE OF CONTENT --- p.V / Chapter CHAPTER 1 - --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 - --- LITERATURE REVIEW --- p.12 / Chapter 2.1. --- Bone transplantation and implant --- p.12 / Chapter 2.1.1. --- "Bone graft, implants and their derivatives" --- p.12 / Chapter 2.1.1.1. --- Survival of donor cells --- p.12 / Chapter 2.1.1.2. --- Osteoclasts --- p.13 / Chapter 2.1.1.3. --- Revascularization --- p.14 / Chapter 2.1.1.4. --- Differentiation of new bone cells by induction --- p.15 / Chapter 2.1.2. --- Phases of bone graft incorporation --- p.15 / Chapter 2.2. --- Preparation and properties of calcium phosphate biomaterials --- p.21 / Chapter 2.2.1. --- Drying and sintering of wet slurries --- p.22 / Chapter 2.2.2. --- Crystal and chemical factors --- p.24 / Chapter 2.2.3. --- Factor governing bioresorbability --- p.25 / Chapter 2.2.4. --- Mineral composition --- p.30 / Chapter 2.2.5. --- Mechanical property of calcium phosphate ceramics --- p.31 / Chapter 2.2.6. --- Basic biologic profile of calcium phosphate implant material --- p.33 / Chapter 2.2.7. --- Osteoinduction --- p.44 / Chapter CHAPTER 3 - --- MATERIALS AND METHODS --- p.46 / Chapter 3.1. --- Animal study --- p.46 / Chapter 3.1.1. --- Animal selection --- p.46 / Chapter 3.1.2. --- Material --- p.46 / Chapter 3.2. --- Methods --- p.46 / Chapter 3.2.1. --- Operation --- p.46 / Chapter 3.2.2. --- Hystology --- p.46 / Chapter 3.2.3. --- Quantitative analysis method --- p.57 / Chapter CHAPTER 4 - --- RESULTS --- p.59 / Chapter 4.1. --- Cortical bone --- p.59 / Chapter 4.1.1. --- Role of periosteum in bone healing --- p.74 / Chapter 4.2. --- Cancellous bone --- p.75 / Chapter CHAPTER 5- --- DISCUSSION --- p.78 / Appendix --- p.86 / References --- p.88

A number of growth factors and cytokines involved in the local regulation of bone remodelling are either synthesised by osteoblasts or have osteoblasts as their target. These include the RANK-L/OPG system, the gp130 cytokine family, including IL-6, and insulin like growth factors. In addition, aberrant cytokine signalling is strongly linked with pathological states characterised by increased bone resorption, including osteoporosis and renal osteodystrophy. The range of action and potency of these osteotropic cytokines requires that their actions are tightly regulated. Amongst such potential control mechanisms are the suppressors of cytokine signalling (SOCS), the presence and role of which in bone has not been studied in detail. The aim of this thesis was (i) to examine the direct effect of uraemia on cytokine release in human osteoblastic cells; (ii) to determine if the regulatory SOCS genes are expressed in these cells and, if so, (iii) to characterise their functional significance. In initial studies, osteoblastic cells were cultured in media containing sera from either healthy volunteers or haemodialysis treated chronic kidney disease patients. Concentrations of OPG and IL-6 were then measured in harvested supernatants. Additionally, individual serum samples collected prior to, and during, a haemodialysis (HD) session were assayed for IL-6, IL-1β and soluble IL-6 receptor (sIL-6R). HD patients had significantly higher concentrations of IL-6 than normal subjects, but there were no significant differences in either IL-1β or sIL-6R. These concentrations did not change significantly during HD. There were no differences in OPG production by osteoblastic cells after exposure to either normal or uraemic serum. Incubation with untreated sera from normal subjects increased IL-6 production by ~6-fold above control, whereas sera from uraemic subjects increased it only ~2-3-fold. HD did not restore the capacity of uraemic serum to augment IL-6 release to the same degree as normal serum. Further work examined a variety of osteotropic stimuli for their ability to induce SOCS1- 3 and CIS expression in human osteoblastic cells. The utility of both conventional RTPCR and fluorescence-based kinetic real time PCR for this purpose are compared. These SOCS were found to be expressed constitutively and could be induced to a variable degree by relevant growth factors. In general, the temporal pattern of SOCS expression was consistent with a negative feedback function. Potential functionality was explored following transfection with SOCS1 and SOCS3 plasmid DNA. Significantly enhanced IL-6 secretion was found in both the basal and stimulated state, whilst OPG production was enhanced only in the latter. Function was also studied in the context of osteoblastic apoptosis, the regulation of which is highly relevant to skeletal disease. Initial experiments developed a framework for subsequent studies: serum starvation for 24h produced reproducible cell death that could be attenuated in a dose dependent manner by IGF-I. SOCS1 and SOCS3 overexpression had limited influence on osteoblast survival, whereas gene knock down experiments using siRNA indicated that IL-1β-induced cell death is mediated differentially, depending on the type of cell death involved. SOCS1 and SOCS3 are involved in the apoptotic cascade, while IL-1β-induced necrosis appears to be independent of SOCS3. Collectively these studies demonstrate that the augmentation of IL-6 production by osteoblastic cells after exposure to normal serum is greater than after uraemic serum. HD does not correct this disparity; perhaps indicating a non-dialysable inhibitor of IL-6 release is involved in the dysregulated bone turnover of uraemic patients. Further work establishes the constitutive presence of the SOCS family in human osteoblastic cells, as well as their transient inducibility by key osteotropic stimuli. Several novel aspects of SOCS function, including influence on IL-6 and OPG production and involvement within apoptotic pathways are demonstrated.

Prostate cancer (PCa) is the most common cancer among men in Western developed countries. While the majority of PCa diagnosed by PSA screening are indolent, advanced and metastatic disease has a significant mortality and morbidity. Bone metastases are extremely common in PCa and identification of bone metastasis associated genes may provide insights into PCa progression and assist in finding new drug targets. However, the genetic study of bone metastases is very limited due to the difficulty of sampling. We performed genome-wide analysis of six fresh frozen PCa bone metastases. We found several alterations commonly present in advanced PCa, including gains at: 1q32.1, broad gains of 8q (MYC, NCOA2), 9q33.2-34.3, 11q13.1-14.1 (CCND1), 12q24.23-24.31, 16p13.3, 16p12.1-11.2 and Xq12-13.1 (AR) as well as losses at: 5q11.1-22.1, 5q14.3-23.1, 6q14.1-22, 8p23.2-p21, 13q13.2-31.1 (RB1), 17p13.1-12 (TP53) and 18q11.1-22.3. Two cases also showed PTEN loss and one sample had deletion indicative of TMPRSS2-ERG fusion. For downstream analysis we concentrated on CCND1 oncogene at 11q13 and FBXL4 at 6q16 as potential drivers of these genomic changes. Using fluorescence in situ hybridisation we found common CCND1 gain and FBXL4 loss in PCa bone metastases (54.5%, 12/22 and 47.8%, 11/23, respectively), much less frequent in primary tumours (7%, 10/142 and 13.8%, 20/145, respectively) and absent in BPH cases (0/55). The expression levels of cyclin D1 protein, coded by CCND1 correlated with CCND1 copy number gain (p < 0.0001) and were higher in metastatic tumours than in primary PCa (p = 0.015), confirming cyclin D1 involvement in advanced PCa. Presence of FBXL4 loss in early stage primary PCa strongly correlated with current PCa prognostic markers and with worse patient survival. Therefore, we propose that FBXL4 may be a tumour suppressor gene in prostate, whose loss in early PCa could be indicative of more aggressive disease. Using in vitro experiments we demonstrated that FBXL4 regulates cells motility and invasion. We confirmed that ERLEC1, an ER lectin involved in ER stress response pathway is a degradation target of FBXL4. As activation of ER stress response pathway is linked to enhanced cell migration and invasion, loss of FBXL4 could be one of the mechanisms by which cancer cells increase their efficiency to respond to stress and to escalate their metastatic potential through stabilisation of ERLEC1. Further studies of FBXL4 - ERLEC1 axis are necessary to establish how they contribute to PCa progression. This knowledge can potentially help to develop novel targeted therapies for aggressive disease harbouring FBXL4 abnormalities.

Shekhar Madhuker Kumta. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 175-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

骨科醫生面臨著老年婦女的骨修復受損或者癒合延遲的挑戰，這使得康復過程變長，甚至引發高死亡率。至今為止，臨床上仍然沒有促進老年骨癒合的滿意治療方法，因此亟需其他治療策略。骨癒合重現了胚胎後的骨骼發育過程。直接由骨外膜成骨（膜內骨化）以及通過軟骨介質成骨（軟骨內骨化）是骨癒合中的兩個重要過程。 雌激素受體β（ERβ基因敲除雌性小鼠的研究表明ERβ信號通路在骨骼發育過程中同時參與了抑制膜內骨化和軟骨內骨化這兩個過程。臨床活檢的資料顯示，在絶經後婦女的骨痂中，ERβ陽性的增生軟骨細胞數量增加。然而，ERβ在正常和老年骨癒合的作用還沒有研究。 / 本研究通過下述部分檢查了ERβ在正常和老年骨癒合的作用，以及其將來的藥物應用：1) 建立一個以膜內骨化為主的骨癒合模型。2) 通過連個骨癒合模型，檢查ERβ在正常骨癒合中的作用。3) 檢查ERβ在老年骨癒合中的作用並檢查ERβ拮抗劑PHTPP 對老年骨癒合的潛在藥物療效。 / 實驗1是建立一個以膜內骨化為主的骨癒合模型。以前建立的小鼠股骨中段骨折模型是軟骨內骨化為主的骨癒合模型。由於技術難度，該模型可重複性不高，而且其金屬內固定器會造成金屬偽影，進而不能應用高解析度微焦點CT跟蹤觀察的技術。為了檢查ERβ在膜內骨化中的作用，並且應用微焦點CT跟蹤觀察技術，我們首先建立了一個小鼠鑽孔缺損模型。該實驗同時也確認了去勢誘導的骨質疏鬆小鼠相比正常小鼠，在鑽孔缺損模型中骨癒合受阻。 / 實驗2檢驗了阻斷ERβ能促進正常骨癒合的假設。本實驗應用ERβ基因敲除小鼠，在兩個模型中檢驗了實驗假設。第一個是傳統的小鼠股骨中段骨折模型，第二個是由實驗1建立的鑽孔缺損模型。兩個模型都證實ERβ基因敲除小鼠骨癒合和野生型小鼠相比，早期的血管新生和中期的礦化有所增強，末期的骨癒合沒有明顯差異。 / 實驗3 進一步研究ERβ在老年骨癒合中的作用。實驗應用老年小鼠股骨中段骨折模型，比較ERβ基因敲除小鼠和野生型小鼠之間的癒合過程。結果顯示ERβ基因敲除小鼠骨癒合和野生型小鼠相比，早期的血管新生，中期的礦化以及末期的力學性能都有所增強。該結果預示阻斷ERβ能作為另一種治療老年骨折癒合的治療策略。同時，我們也檢測了ERβ的拮抗劑PHTPP（4 - [2 - 苯基- 5,7 -二（三氟甲基）吡唑並[1,5 - A]嘧啶3 - 基]苯酚, 在老年骨癒合中的治療效果。 通過比較用藥組小鼠與安慰劑組小鼠的骨癒合品質，顯示PHTPP治療小鼠血管新生，骨痂礦化和最終的力學性質均優於對照安慰劑組小鼠。 / 綜上所述，本研究描述了ERβ在正常和老年骨癒合中的作用。骨癒合的關鍵過程包括血管新生，膜內骨化以及軟骨內骨化在阻斷ERβ後都得到增強，從而加快正常骨和老年骨的骨痂形成，礦化並增強力學性質。ERβ的拮抗劑PHTPP在老年小鼠骨折模型中能促進骨癒合。本研究提出了一個新的骨癒合治療策略，並為將來的臨床實驗提供了堅實的基礎。 / Orthopaedic surgeons are challenged by impaired or delayed bone healing in elderly women, which requires prolongation of rehabilitation process or even induces high mortality. Up to date, there are no satisfactory therapeutic modalities for promoting aged bone healing clinically, and alternative therapeutic stratagem is therefore desirable. Bone healing recapitulates postnatal bone development. Direct periosteam-dependent bone formation (intramembranous ossification) and the formation of bone through a cartilage intermediate (endochondral ossification) are the two important processes during bone healing. Evidences from Estrogen Receptor β (ERβ), gene knockout female mouse studies have demonstrated that ERβ signaling participates in inhibiting both intramembranous and endochondral ossification during bone development. Clinical biopsy data demonstrated that the number of ERβ positive proliferative chondrocytes within fracture callus was increased in postmenopausal women. However, the role of ERβ in normal and aged bone healing is not examined yet. / This study examined role of ERβ in normal and aged bone healing and the future pharmaceutical application though the following part: 1) Establish an intramembranous ossification-dominant bone healing model. 2) Examine the role of ERβ in normal bone healing though two models. 3) Examine the role of ERβ in aged bone healing and investigate the potential therapeutical efficacy of an ERβ antagonist PHTPP in aged bone healing. / Study I was to establish an intramembranous ossification dominant bone healing mouse model. Previous available mouse femoral shaft fracture model was a endochondral ossification dominant bone healing model. This model was technically difficult to generate high reproducibility and the inside metal stabilization devices prevented the application of high-resolution in vivo micro-CT monitoring due to the metal artifact. In order to examine the role of ERβ in intramembranous ossification and apply the micro-CT monitoring technique, a drill-hole defect mouse model was developed. The study also confirmed bone healing was impaired in mice with ovariectomy -induced osteoporosis in drill-hole defect model. / Study II was to test the hypothesis that blockade of ERβ could promote normal bone healing. ERβ knockout mice were employed in this study and the hypothesis was examined in two models, the first is the traditional mouse femoral shaft fracture model, and the second is the drill-hole defect model that was developed in study I. Both models demonstrated that the bone healing in ERβ knockout mice was enhanced in the early stage of neovascularization and the middle stage of ossification but not by the end of healing compare to the wild type mice. / Study III was designed to further investigate the role of ERβ in aged bone healing. Femoral shaft fracture model was created in aged mice. The healing process was compared between the ERβ knockout mice and wild type mice. The results demonstrated that ERβ knockout mice was enhanced in the early stage of neovascularization, the middle stage of ossification and end stage of mechanical strength. The findings implied blockade of ERβ can be considered as another therapeutic strategy for aged fracture healing. PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl] phenol), an ERβ antagonist, was employed in aged mice femoral shaft fracture model. The bone healing quality of treated mice was compared with that of the vehicle control mice. It showed PHTPP treated mice had enhanced neovascularization, callus ossification and finally better mechanical properties than vehicle mice. / The present study depicted the role of ERβ in normal and aged bone healing. Key processes including neovascularization, intramembranous and endochondral ossification were all enhanced by blockade of ERβ, which led to fast callus formation, mineralization in normal bone and better mechanical properties in aged bone. ERβ antagonist PHTPP could promote aged bone healing in mouse osteotomy model. This study raised an alternative therapeutic stratagem for bone healing and provided solid basis for future clinical trials. / 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. / He, Yixin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 147-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 中文摘要 --- p.iv / PUBLICATIONS AND AWARDS --- p.vi / ACKNOWLEDGEMENTS --- p.xi / TABLE OF CONTENTS --- p.xii / LIST OF ABBREVIATIONS --- p.xvi / LIST OF FIGURES --- p.xviii / LIST OF TABLES --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION AND LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Fracture and Bone Healing --- p.2 / Chapter 1.1.1 --- Epidemiology and Impacts of Fractures --- p.2 / Chapter 1.1.2 --- Current Management and Limitations --- p.3 / Chapter 1.1.3 --- Bone Structures --- p.5 / Chapter 1.1.4 --- Bone Healing --- p.7 / Chapter 1.1.5 --- Aged Bone Healing --- p.12 / Chapter 1.1.6 --- Enhancements of Bone Healing --- p.17 / Chapter 1.2 --- Estrogen and Estrogen Receptors --- p.19 / Chapter 1.2.1 --- Estrogen Receptors α and β --- p.19 / Chapter 1.2.2 --- Molecular Actions of Estrogens --- p.20 / Chapter 1.2.3 --- Estrogen receptors in bone homeostasis --- p.24 / Chapter 1.3 --- Hypothesis --- p.28 / Chapter 1.4 --- Study Plan and Objectives --- p.32 / Chapter 1.4.1 --- Bone Healing Models --- p.32 / Chapter 1.4.2 --- Study Outline --- p.32 / Chapter 1.5 --- Figures and Tables --- p.34 / Chapter CHAPTER 2 --- ESTABLISHMENT OF DRILL-HOLE DEFECT HEALING MODEL IN MICE --- p.39 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.1.1 --- Limitations in currently available mouse models of osteoporotic bone healing --- p.40 / Chapter 2.1.2 --- Creation of a drill-hole defect at the mid-diaphysis of the femur for in vivo monitoring of bone healing in mice --- p.40 / Chapter 2.2 --- Materials and Methods --- p.43 / Chapter 2.2.1 --- Experimental animals --- p.43 / Chapter 2.2.2 --- Surgical protocol and experimental design --- p.43 / Chapter 2.2.3 --- Micro-CT analysis of intact femur --- p.44 / Chapter 2.2.4 --- In vivo micro-CT analysis of new bone formation in the drill-hole site --- p.45 / Chapter 2.2.5 --- Micro-CT-based angiography --- p.45 / Chapter 2.2.6 --- Histological examination --- p.46 / Chapter 2.2.7 --- Immunohistochemistry --- p.46 / Chapter 2.2.8 --- Quantitative real-time PCR --- p.47 / Chapter 2.2.9 --- Analysis of bone formation and resorption markers --- p.47 / Chapter 2.2.10 --- Mechanical testing --- p.48 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter 2.3 --- Results --- p.51 / Chapter 2.3.1 --- Confirmation of osteoporotic bone prior to generation of a drill-hole defect --- p.51 / Chapter 2.3.2 --- General observation of mice following drill-hole surgery --- p.51 / Chapter 2.3.3 --- In vivo micro-CT analysis of new bone in the drill-hole site of mouse femurs --- p.51 / Chapter 2.3.4 --- In vivo micro-CT analysis of new bone in drill-hole sites is highly reproducible --- p.52 / Chapter 2.3.5 --- Micro-CT angiography --- p.52 / Chapter 2.3.6 --- Histological observation of bone healing --- p.53 / Chapter 2.3.7 --- Immunohistochemical analysis of ER expressions during bone healing --- p.54 / Chapter 2.3.8 --- Quantitative real-time PCR analysis of gene expression during bone healing --- p.54 / Chapter 2.3.9 --- Analysis of bone formation and resorption markers during bone healing --- p.54 / Chapter 2.3.10 --- Mechanical testing of femurs from Sham and OVX mice --- p.55 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Bone healing with dominant intramembranous ossification --- p.56 / Chapter 2.4.2 --- Impaired osteoporotic bone healing --- p.57 / Chapter 2.4.3 --- Reproducibility of the in vivo micro-CT method for analysis of bone healing --- p.58 / Chapter 2.4.4 --- Dysregulated expression of estrogen receptors and bone healing in OVX mice --- p.59 / Chapter 2.4.5 --- Study limitations --- p.60 / Chapter 2.4.6 --- Conclusions --- p.60 / Chapter 2.5 --- Figures and Tables --- p.61 / Chapter CHAPTER 3 --- ROLE OF ERβ IN NORMAL BONE HEALING --- p.72 / Chapter 3.1 --- Introduction --- p.73 / Chapter 3.2 --- Materials and Methods --- p.75 / Chapter 3.2.1 --- Part I Study --- p.75 / Chapter 3.2.1.1 --- Experimental animals --- p.75 / Chapter 3.2.1.2 --- Fracture model and experimental design --- p.75 / Chapter 3.2.1.3 --- Radiographic Analysis --- p.76 / Chapter 3.2.1.4 --- Micro-CT-based angiography --- p.76 / Chapter 3.2.1.5 --- Micro-CT analysis of callus --- p.77 / Chapter 3.2.1.6 --- Histological examination --- p.78 / Chapter 3.2.1.7 --- Dynamic Bone histomorphometric analysis --- p.78 / Chapter 3.2.1.8 --- Mechanical testing --- p.79 / Chapter 3.2.1.9 --- Quantitative real-time PCR --- p.80 / Chapter 3.2.1.10 --- Analysis of bone formation and resorption markers --- p.80 / Chapter 3.2.1.11 --- Statistical analysis --- p.81 / Chapter 3.2.2 --- Part II Study --- p.81 / Chapter 3.2.2.1 --- Experimental animals and design --- p.81 / Chapter 3.2.2.2 --- Evaluation protocols --- p.82 / Chapter 3.2.2.3 --- Statistical analysis --- p.82 / Chapter 3.3 --- Results --- p.83 / Chapter 3.3.1 --- Part I Study --- p.83 / Chapter 3.3.1.1 --- Radiographic Analysis --- p.83 / Chapter 3.3.1.2 --- Micro-CT angiography --- p.83 / Chapter 3.3.1.3 --- Micro-CT analysis of callus --- p.83 / Chapter 3.3.1.4 --- Histological and dynamic histomorphometric analysis --- p.84 / Chapter 3.3.1.5 --- Mechanical testing of the callus --- p.85 / Chapter 3.3.1.6 --- Quantitative real-time PCR analysis of gene expression --- p.85 / Chapter 3.3.1.7 --- Analysis of bone formation and resorption markers during bone healing --- p.85 / Chapter 3.3.2 --- Part II Study --- p.86 / Chapter 3.3.2.1 --- In vivo micro-CT analysis of new bone in the drill-hole site of mouse femurs --- p.86 / Chapter 3.3.2.2 --- Micro-CT angiography --- p.87 / Chapter 3.3.2.3 --- Histological observation of bone healing --- p.87 / Chapter 3.3.2.4 --- Quantitative real-time PCR analysis of gene expression --- p.88 / Chapter 3.3.2.5 --- Analysis of bone formation and resorption markers during bone healing --- p.88 / Chapter 3.3.2.6 --- Mechanical testing of femurs from WT and KO mice --- p.88 / Chapter 3.4 --- Discussion --- p.90 / Chapter 3.4.1 --- Angiogenesis --- p.90 / Chapter 3.4.2 --- Fracture Healing --- p.91 / Chapter 3.4.3 --- Estrogen receptor β and endochondral and intramembranous ossification --- p.93 / Chapter 3.4.4 --- Estrogen receptor β in aged bone --- p.94 / Chapter 3.4.5 --- Conclusions --- p.94 / Chapter 3.5 --- Figures and Tables --- p.95 / Chapter CHAPTER 4 --- ROLE OF ERβ AND ITS ANTAGONIST PHTPP IN AGED BONE HEALING --- p.113 / Chapter 4.1 --- Introduction --- p.114 / Chapter 4.2 --- Materials and Methods --- p.116 / Chapter 4.2.1 --- Experimental animals --- p.116 / Chapter 4.2.2 --- Fracture model and experimental design --- p.116 / Chapter 4.2.3 --- Radiographic Analysis --- p.117 / Chapter 4.2.4 --- Micro-CT-based angiography --- p.118 / Chapter 4.2.5 --- Micro-CT analysis of callus --- p.118 / Chapter 4.2.6 --- Histological examination --- p.119 / Chapter 4.2.7 --- Dynamic Bone histomorphometric analysis --- p.120 / Chapter 4.2.8 --- Mechanical testing --- p.120 / Chapter 4.2.9 --- Quantitative real-time PCR --- p.121 / Chapter 4.2.10 --- Analysis of bone formation and resorption markers --- p.122 / Chapter 4.2.11 --- Statistical analysis --- p.122 / Chapter 4.3 --- Results --- p.123 / Chapter 4.3.1 --- Radiographic Analysis --- p.123 / Chapter 4.3.2 --- Micro-CT angiography --- p.123 / Chapter 4.3.3 --- Micro-CT analysis of callus --- p.123 / Chapter 4.3.4 --- Histological and dynamic histomorphometric analysis --- p.124 / Chapter 4.3.5 --- Mechanical testing of the callus --- p.125 / Chapter 4.3.6 --- Quantitative real-time PCR analysis of gene expression during fracture healing --- p.125 / Chapter 4.3.7 --- Analysis of bone formation and resorption markers during bone healing --- p.126 / Chapter 4.4 --- Discussion --- p.127 / Chapter 4.4.1 --- Angiogenesis --- p.127 / Chapter 4.4.2 --- Fracture Healing --- p.128 / Chapter 4.4.3 --- Estrogen receptor β and endochondral ossification --- p.129 / Chapter 4.4.4 --- ERβ antagonist PHTPP --- p.130 / Chapter 4.4.5 --- Conclusions --- p.130 / Chapter 4.5 --- Figures and Tables --- p.131 / Chapter CHAPTER 5 --- STUDY LINITATIONS, FURTHER RESEARCH AND CONCLUDSIONS --- p.142 / Chapter 5.1 --- Limitations --- p.143 / Chapter 5.1.1 --- Bone healing model --- p.143 / Chapter 5.1.2 --- Estrogen receptors and transgenic mouse --- p.143 / Chapter 5.1.3 --- ERβ antagonist PHTPP --- p.144 / Chapter 5.2 --- Further Research --- p.144 / Chapter 5.2.1 --- ERβ signaling --- p.144 / Chapter 5.2.2 --- Preclinical Trial --- p.145 / Chapter 5.3 --- Conclusions --- p.146 / BIBLIOGRAPHY --- p.147

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

In conclusion, the present study firstly addressed the relationship between graft incorporation and peri-graft bone quality and quantity after ACL reconstruction in a rabbit model. The findings suggested that the non-invasive measurement of peri-graft bone would be useful to predict graft incorporation. Peri-graft bone loss was region-specific after surgery, which might be associated with stress shielding in the specific region after tunnel creation. The use of Brushite CPC might be a promising way to augment peri-graft bone and enhance graft incorporation. (Abstract shortened by UMI.) / In the fourth part, brushite CPC was successfully applied to augment the peri-tendon bone volume and connectivity. It was revealed under mechanical testing that the ultimate strength and stiffness of graft fixation in bone tunnel on experimental side was higher than that of the control by 117% and 102% respectively at 6 weeks, postoperatively (p&lt;0.05 for both). The use of brushite CPC caused a paradigm shift in failure mode from intra-tunnel to intra-articular portion at 12 weeks postoperatively (p=0.013). / Keywords. anterior cruciate ligament reconstruction, bone mass, microarchitecture, tendon-to-bone healing, graft incorporation, brushite calcium phosphate cement / The first part of this thesis compared the histological characteristics of T-B healing interface tissue in femoral and tibial tunnels following ACL reconstruction in rabbits. Results revealed that less cartilaginous interface tissue was formed in tibial tunnel than in femoral tunnel. Such cartilaginous tissue was gradually mineralized during reestablishment of a direct T-B integration. T-B healing in tibial tunnel was inferior to that in femoral tunnel. The disparity of T-B healing in various osseous milieus suggested the potential association in between. / The second part of this original work further explored the numeric relationship between the strength of T-B attachment and peri-graft bone mass and connectivity. Results of Micro-computed tomography (micro-CT) showed that peri-graft bone mass and connectivity was significantly lower on tibial side than those on femoral side. It was found under biomechanical evaluation that grafted tendon was prone to be pulled out from tibial tunnel with the bone attachment; the weakest point of the complexes shifted from the healing interface at time zero to peri-graft bone at week 6 after operation. With reverse of peri-graft bone at week 12 postoperatively, the weakest point shifted to intra-osseous tendinous portion. The stiffness of graft fixation correlated with peri-graft BV/TV (r2=0.68, p=0.001) and connectivity (r2=0.47, p=0.013) at week 6 after operation. / The third part addressed the changes of peri-graft bone in spatial and temporal manners using high resoluation multiple-slice peripheral quantitative computed tomography (pQCT) and micro-CT. Under pQCT evaluation, a decrease in BMD was present in specific regions, medial region of femoral tunnel by 26% and posterior and lateral regions of tibial tunnel by 22% and 42%, respectively at week 12 postoperatively than the baseline (p&lt;0.05 for all). It was accompanied by a decrease in trabecular number and increase in trabecular spacing, the shift of plate-like to rod-like trabeculae and loss of anisotropy under micro-CT evaluation. It was echoed by histological findings showing increased osteoclastic activities and poor T-B healing in these specific regions. The postoperative bone loss and associated poor T-B healing was region-specific. / by Wen, Chunyi. / Adviser: Kai-ming Chan. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0217. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 148-168). / 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. / Abstracts in English and Chinese.

Thesis (M Med (Haematology))--University of Limpopo, 2010. / Background. Infection with human immunodeficiency virus (HIV) is associated with a range of haematological abnormalities including: ineffective haematopoiesis, infiltrative disease of the bone marrow, nutritional deficiency and peripheral destruction of blood cells secondary to splenomegaly and immune deregulation. Aim. To review and describe bone marrow abnormalities and associated peripheral haematological abnormalities, in HIV/AIDS patients. Methodology. This is a retrospective study. Data was extracted from DISA, the National Laboratory Health Service Laboratory Information System database at the DGMH Tertiary Laboratory from 2003 to 2007. Medical and laboratory records of 80 HIV positive patients who underwent bone marrow examination for investigation of fever and/or cytopenia were reviewed. All statistical analyses were performed on SAS® Release 9.1.3. Results. Twenty-five patients out of a total of 80 (31.25%), had pancytopaenia. Of the 25, eight (32%) were males and 17 (68%) were females. In this study, pancytopaenia was described as a haemoglobin concentration, granulocyte count and platelet count below normal ranges for age and gender. Among male patients in this study, five (17%) patients had TB out of 30. Among female patients, five (10%) out of 50 patients had TB. The majority of patients with malignancies were males six out of nine (67%). Three of the five patients with non-Hodgkin’s lymphoma (NHL) and all of the patients with multiple myeloma (MM) were males. Conclusions. Haematological abnormalities were present in all patients. Bone marrow involvement by TB was found in 12.5% in the study population. Malignancies were more frequent in males; three patients with NHL, two with MM and one with Kaposi sarcoma (KS). The difference in distribution was not statistically significant (p=0.391002). Recommendations. It is recommended that health education and health promotion focus on the control of biological carcinogenic agents such as EBV, HPV and HHV-8 by routinely testing for these agents and by promotion of positive reproductive behaviour among people living with HIV/AIDS. The use of non-invasive tests will be helpful in our setting where there is high TB prevalence.

Master of Dental Surgery / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Osteoporosis is a growing health concern, characterised by deterioration of bone and increased fracture incidence. Anabolic treatments for reversing bone loss are presently limited. A bone anabolic response was recently reported following deletion of hypothalamic neuropeptide Y2 receptors in mice. In contrast, no discernable bone phenotype was observed in Y4 receptor knockout (Y4-/-) mice, revealing specificity between the Y receptors in their control of bone formation. Studies in this thesis revealed a second anabolic response in the absence of another Y receptor subtype; the Y1 receptor. The potential interaction between the Y1 and Y2- anabolic pathways with each other and with Y4 was investigated through the generation of mouse models lacking multiple Y receptor subtypes. Interestingly, no synergistic elevation in bone volume was observed in Y1-/-Y2-/- double knockout mice, indicative of shared mechanisms of action. In contrast, the synergistic elevation in bone volume of male Y2-/- Y4-/- mice was likely due to additive effects of leptin signalling. Consequentially, potential interaction between Y receptors and leptin was investigated by crossing the Y receptor knockouts onto the leptin deficient ob/ob background, revealing differential responses of the Y receptor pathways to leptin deficiency, with the anabolic response of the Y2-/- model retained in Y2-/-/ob mice but abolished in Y1-/-/ob mice compared to Y1-/-. Differential responses of these two pathways were also revealed following gonadectomy of Y1-/- and Y2-/- mice. Importantly, these studies also demonstrated the ability of the central Y2- anabolic pathway to halt gonadectomy-induced bone loss. Interestingly, cultured stromal cells from germline Y2-/- mice exhibited an enhanced ability to undergo mineralisation and adipocyte differentiation, associated with a greater number of mesenchymal progenitor cells present within the bone of Y2-/- mice, suggesting a potential mechanism for the greater mineralisation of the Y2-/- model in vitro and in vivo. Y1 receptor expression was also detected in stromal cells from wild type mice, but was nearly abolished in Y2-/- mice. Together these findings demonstrate an important therapeutic potential for these pathways in the treatment of osteoporosis and indicate that modulation of Y receptor signalling within the bone microenvironment may alter proportions of mesenchymal progenitor populations with effects on bone formation.