Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

The objective of this research was to evaluate the relationships between greater adiposity and bone development during adolescence. Bone was evaluated from age 11 to 17 years in the Iowa Bone Development Study using peripheral quantitative computed tomography (pQCT). Body composition (fat and lean mass) was estimated by dual energy x-ray absorptiometry (DXA). The first research aim evaluated the associations between greater overall adiposity and subsequent maturation and bone strength in 135 girls and 123 boys. Greater adiposity was defined according to age 8 Body Mass Index (BMI) to categorize participants as overweight (OW) or healthy-weight (HW). Maturation was defined as the age of peak height velocity (PHV). Bone strength was assessed at the radius and tibia (bone strength index, BSI, and strength-strain index, SSI). Differences in bone strength between OW and HW were evaluated with sex-specific multi-level regression models to account for individual growth and correlation between repeated measurements. Analyses were adjusted for centered age (measurement visit age - grand mean age of cohort), change in fat mass, and limb length in Model 1, with additional adjustment for lean mass in Model 2. Analyses were repeated using biological age (visit age - age PHV). BMI was positively associated with age of maturation in girls and boys (p< 0.05). HW versus OW girls had significantly lower BSI and SSI at the radius and tibia (p< 0.05) in Model 1. Results remained significant except for radial BSI in Model 2. HW versus OW boys had significantly lower BSI and SSI (all p< 0.05) at the tibia, but not radius, in Model 1. Significant differences were sustained in Model 2. Analyses were repeated using biological age, which yielded similar results for boys, but reduced parameter estimates were observed in girls, with only tibial SSI significant in Model 2 (p< 0.05). These findings support a stronger role for greater adiposity in the occurrence of earlier maturation and greater bone strength in girls than boys while greater lean mass appeared to play a greater role in boys. The second research aim evaluated associations between abdominal adiposity and bone in 132 girls and 122 boys. Visceral adipose tissue area (VAT, cm2) and subcutaneous adipose tissue area (SAT, cm2) were estimated from DXA scans. Sex-specific analyses evaluated the fat-bone relationship with growth models using biological age as the time variable adjusted for limb length and lean mass. There were no significant associations between bone parameters and VAT or SAT in girls. In boys, greater VAT was associated with lower trabecular bone density (tBMD) and BSI (all p< 0.05) at the tibia, but not radius. Greater VAT and SAT were associated with smaller cortical bone size and thickness (all p< 0.01) at the radius, but not tibia. Analyses limited to overweight participants showed VAT was negatively associated with periosteal circumference at the radius and tibia, cortical bone thickness at the tibia and SSI (all p< 0.05) at the radius in girls. In boys, the results were relatively unchanged for VAT, while SAT was only significantly associated with lower tBMD (p< 0.05) at the tibia. These results suggest the bone-fat relationship may vary depending on adiposity and bone site. The third research aim evaluated the longitudinal association between intramuscular fat and cortical bone at the tibia from age 11 to 17 years in 153 girls and 143 boys. Muscle density (MD) was used to estimate intramuscular fat (IMF). Lower MD indicates greater IMF. The relationships between muscle density and cortical bone parameters were modeled using multi-level regression models adjusted for biological age, limb length and muscle cross-sectional area measured by pQCT. In the adjusted multi-level regression models, MD was positively associated with cortical bone parameters, but only reached statistical significance for BMD, bone mineral content (BMC), bone cross-sectional area, cortical thickness and SSI in girls, while only SSI was significant in boys (all p< 0.05). These results suggest that greater fat content within muscle may be harmful to weight-bearing cortical bone during adolescence. In conclusion, findings from the first aim suggest there are sex- and site-specific differences in the relationship between adiposity and bone during adolescence. Findings from the second and third aims indicate these differences could be explained, in part, by the existence of specific fat depots (abdominal more so than intramuscular fat) that could be harmful to bone and that may be more apparent in boys due to a sex-specific fat distribution pattern that favors accumulation of abdominal rather than peripheral fat.

No description available.

The effect of maternal dietary fats on growth rate and bone development of commercial broilers was examined. Three hundred fifty female chicks were winged banded, weighed and equally divided among six starter pens (1.52 X 3.66m) with litter floors. At 20 wk of age, each pen was fed a basal laying diet supplemented with either 3% chicken fat (CF), soybean oil (SBO) or menhaden oil (MO). Each diet was provided with or without the antioxidant ethoxyquin, producing a total of six dietary treatments. Addition of fats [soybean (SBO), menhaden oil (MO), chicken fat (CF), soybean + antioxidant (SA), menhaden + antioxidant (MA), and chicken + antioxidant (CA)] to the maternal diet altered the tissue and yolk composition of hens to reflect the dietary source. Response variables measured were body weight, tibia weight and length, and breaking strength (stress, force, energy, bone wall, and diameter). Chick tissue from hens fed a MO and MA diet exhibited greater (P<0.01) amounts of DPA (22:5n3), DHA (22:6n3) and total n-3 fatty acids than the remaining dietary treatments. Tissues from chicks fed a SBO and SA diet displayed larger levels of 18:2n6 and total n-6 fatty acids when compared to all other treatments. Male and female chicks from the menhaden type diets (MO and MA) were lighter (P<0.01) during grow out period than from soybean (SBO and SA) and chicken (CF and CA) type diets. Chicks tibiae diameter from CF maternal diet tended to be larger than the MO maternal diet, with significance being noted at d 14 (P<0.01) and 28 (P<0.01). Increases were observed in shear force and stress required to break chick tibia from SBO maternal diet compared to those from the CF and MO maternal diets. The SBO maternal diet stimulates growth rate and bone development and strength of the progeny. (Key words: chickens, bone development, breaking strength, growth rate, fatty acids) / Master of Science

The objective of these studies was to design an optimal nutritional supplement suitable for grazing horses using fat and fiber to replace the grain and molasses in the traditional sweet feed. Thoroughbred mares and foals grazing bluegrass/clover pastures were used in these studies, twenty mares and their foals in 1994 to 1995, and twenty mares and foals in 1995 to 1996. Seasonal variation in pasture was examined, and the need for supplementation of nutrients and fibers was assessed. The nutritional status of grazing mares, foals, weanlings and yearlings, fed either a starch and sugar supplement (SS) or a fat and fiber supplement (FF), was examined using growth measurements, radiographic bone evaluations, milk composition and glucose tolerance tests. These studies suggest that fiber may be an important component of an ideal supplement for improved grass/legume pastures. Seasonal variation in pasture indicated an increase in hydrolyzable and rapidly fermed carbohydrates during periods of rapid growth. The FF supplement may have buffered seasonal changes and the increased hydrolyzable carbohydrate content in rapidly growing pasture, as evidenced by smoother growth curves in the yearlings. Young horses, after weaning until the following May, had lower estimated bone mineral content when fed the FF supplement. The lower bone mineral content in the FF supplemented horses may have been due to decreased absorption of calcium or metabolic and hormonal changes associated with adaptation to the different energy sources in the supplements. Milk composition of FF supplemented mares was influenced in ways likely to improve foal health. The FF supplemented mares had enhanced linoleic acid content, which may reduce the risk of gastric ulcers in foals, and increased immunoglobulin G concentration, which may enhance passive immunity. The carbohydrate status of mares, as assessed by glucose tolerance tests, indicated a slower glucose clearance that could be a metabolic adaptation of the mares to the SS and FF supplements. / Ph. D.

We evaluated coxofemoral joints from museum specimens of: Vulpes lagopus; Vulpes vulpes; Vulpes velox; Nyctereutes procyonoides; Urocyon cinereoargenteus; Aenocyon [Canis] dirus; Canis latrans; Canis lupus lupus; Canis lupus familiaris; C. l. familiaris × latrans; and Canis dingo. Acetabular components included: fossa; articular surface; medial and lateral articular margins; and periarticular surfaces. Acetabular components variably revealed: osteophyte-like features; varying appearance of articular margin rims (especially contour changes); rough bone surfaces (especially fossa and articular surface); and surface wear. Proximal femoral components included: articular surface; articular margin; periarticular surfaces; and joint capsule attachment. Femoral components variably revealed: rough bone surface; bone loss; articular margin osteophyte-like features; caudal post-developmental mineralized prominence; and enthesophytes along the joint capsule attachment. Non-metric multidimensional scaling was used to analyze right-left asymmetric relationships between observed traits, across taxa. Significantly different acetabular trait asymmetry involved only C. latrans-C. l. familiaris; V. vulpes-N. procyonoides, and U. cinereoargenteus-N. procyonoides. There were no significant lateralized differences in proximal femoral traits involving modern canids, ancient and modern C. l. familiaris, or modern vulpines. Thus, the observations were strongly bilateral. We hypothesized high similarity of traits across taxa. The data confirm the hypothesis and strongly suggest broad and deep morphological and mechanistic conservation that almost certainly pre-existed (at least) all modern canids. Further zoological studies are needed to evaluate phylogenic implications in greater detail.

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

by Tang Shengping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 217-244). / 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. / Abstracts in English and Chinese.

骺軟骨板是位於長骨骨骺的一個軟骨結構，其主要功用為使骨骼延伸生長。 若人類骺軟骨板基因出現障礙，骨骼生長往往會受到嚴重的影響，使骨骼變得短小、畸形，造成殘障。此外，一些後天的系統性失調也會損害骺軟骨板的正常運作，導致矮小症。現今常用來醫治骨骼生長障礙的包括重組的人類生長荷爾蒙。然而，它的功效並非顯著，亦帶來很多的副作用；因此，我們希望能尋求更好的醫治方法。 / 近有的研究結果顯示，很多的內分泌及分泌因子能夠刺激骺軟骨板進行軟骨增生。但是，研究者卻往往未能將這些因子轉化及運用作醫療用途，原因是它們通常都是局部性地於骺軟骨板產出及發生效用。倘若以上提及的生長因子能給骺軟骨板的靶子蛋白鏈準確地被帶往骺軟骨板，那麼這些因子便能有效地被利用作治療用途，而其效能亦會給大大提高，副作用也會被減低。因此，我們現在進行研究的首要目標為於採用噬菌體展示和酵母展示方法， 尋找那些能認辨出骺軟骨板的靶子蛋白鏈及單鏈抗體。 / 於噬菌體展示庫裡，我們找出了兩條能認辨出小鼠骨骼軟骨細胞的靶子蛋白鏈 － C1 (RLDPTSYLRTFW) 和 C19 (HDSQLEALIKFM)。然而於體外測試實驗中， 它們對軟骨細胞及細胞外基質只擁有中度的親近性和特異性。此後，於酵母展示庫裡，我們亦發現三條可認辨某骺軟骨板蛋白的單鏈抗體 － 它們的親近性甚高 (達至1 nM)，而其對軟骨組織的特異性也為良好 (它們只認辨軟骨組織，但卻沒能認辨出其他的軟組織，包括腦、心臟、肝臟、肺臟、腎臟、脾臟、小腸及肌肉)。 其後，於小鼠胚胎免疫染色測試實驗中，這些單鏈抗體只亦選擇地認辨軟骨組織。再者，當這些單鏈抗體被注射入小鼠的靜脈中，它們也會準確地停留在軟骨組織裡，顯示出其特異性於體內測試實驗中亦存在。 / 總括而言，利用噬菌體展示和酵母展示方法，我們發現了一些能認辨出骺軟骨板的靶子蛋白鏈及單鏈抗體。這些單鏈抗體擁有對軟骨組織非常高的親近性和特異性。我們預計，若然將這些單鏈抗體和內分泌及分泌因子連結在一起，它們或能成為醫治骨骼生長障礙的新方法。 / The growth plate is a specialized cartilage structure at the ends of long bones that is responsible for bone elongation. Many human genetic disorders of the growth plate impair skeletal growth, often resulting in bones that are severely short and malformed, causing serious disability. Many acquired systemic disorders also impair growth plate function, causing short stature. Currently, recombinant human growth hormone is used to treat growth disorders, but it has limited efficacy for severe diseases and causes significant adverse effects. / Recent studies have identified many endocrine and paracrine factors that are capable of promoting chondrogenesis at the growth plate. However, the development of these molecules into effective therapies has been hampered by their action mechanism; they are produced locally and act locally in the growth plate and thus fail to lend themselves to systemic therapeutic approaches. We envisioned that, if these growth factors could be linked to growth plate-targeting peptides or polypeptides and then administered systemically, these molecules might provide a better treatment strategy for growth disorders because targeting might augment the therapeutic effect on the growth plate but diminish undesirable effects on non-target tissues. Toward this goal, we sought to identify peptides and antibody fragments that bind to cartilage tissue using phage display and yeast display technologies. / We used a phage display library that expressed linear peptides of 12 random amino acids on the phage surface and then selected for binding to murine primary cultured chondrocytes. This approach successfully identified two peptides, namely C1 (RLDPTSYLRTFW) and C19 (HDSQLEALIKFM), which exhibited moderate binding affinity and specificity to chondrocytes and extracellular matrix in vitro. We also used a yeast display library to identify three single-chain variable(scFv) antibody fragments that bound strongly to a growth plate-specific protein(EC50 values less than 1 nM). These antibody fragments demonstrated specific binding in vitro to homogenates of cartilage tissues, but not homogenates of brain, heart, liver, lung, kidney, spleen, small intestine, or muscle. Moreover, they also exhibited tissue-specific binding to cartilage structures in sections of mouse embryos. When these purified antibody fragments were injected intravenously in mice, they localized to cartilage and were not detectable in other tissues, including brain, heart, liver, lung, kidney, spleen, small intestine, or muscle, indicating that the antibody fragments were capable of specifically targeting cartilage tissue in vivo. / In conclusion, we employed phage display and yeast display methods to identify peptides and antibody fragments that bind to cartilage tissues. The selected antibody fragments showed particularly high binding affinity and specificity to cartilage. Conjugating these antibody fragments to endocrine and paracrine signaling molecules has the potential to provide targeted therapy for growth plate disorders. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cheung, Sao Fong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 89-102). / Abstracts also in Chinese.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.