Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Insulin is a mitogen for many cells and commonly signals through the classical, mitogenic Raf- MEK-ERK or metabolic PB-kinase pathways. Insulin deficiency or type I diabetes causes severe osteopenia. Obese patients with type II diabetes or insulin resistance, a disease associated with defective insulin signaling pathways and high levels of circulating insulin, have increased or normal bone mineral density. The question of whether hyperinsul inemia preserves bone mass is frequently raised. However, there is still a lot of controversy on the role of insulin as an osteoanabolic agent and this question still remains unanswered. A critical role for insulin signaling in bone building osteoblasts has recently been demonstrated with IRS-l knock-out mice. These mice developed low-turnover osteopenia due to impaired proliferation and differentiation, stressing the importance of osteoblastic IRS-l for maintaining normal bone formation. In the present study it was found that insulin does function in vitro as an osteoblast mitogen. This was illustrated in three relatively immature osteoblast (MBA-15.4, -15.6 mouse and MG- 63 human) cell lines, which responded to insulin with significant increases in proliferation. In the MBA -15.4 preosteoblasts insulin stimulation of proliferation was comparable to the welldescribed mitogen, TPA. The UMR-I06 cell line expresses markers of differentiated osteoblasts, and was much less responsive to insulin treatment. The difference in proliferative potential may be due to differences between spontaneously transformed cell lines, or the stage of cell differentiation. UOI26, a MEKI/2 inhibitor and wortmannin, a PB-kinase inhibitor, were used to investigate the pathway used by insulin to signal and activate ERK and osteoblast proliferation. In MBA-15.4 mouse preosteoblasts, GF-containing FCS was completely dependent on MEK for DNA synthesis. In contrast, in both MBA-15.4 and more mature MBA-15.6 osteoblasts, insulininduced proliferation was resistant to the inhibitors alone or in combination. Higher MEKinhibitor concentrations had no effect, and proliferation was also increased by the inhibitors in several experiments. This indicated that the classical, insulin mitogenic pathway was not involved in MBA-15.4 proliferation. Wortmannin had no effect on either insulin- or 20% FCSstimulated proliferation, but inhibited activation of Akt/PKB, the metabolic downstream target of PI3-kinase. Insul in signal ing to ERK was both MEK-and PI3-kinase- dependent, but this had no effect on proliferation. In contrast, FCS-stimulated ERK activation and proliferation was almost completely dependent on MEK-ERK activation. Proliferative signaling in the MG-63 human osteoblastic cell line in response to insulin was partially dependent on MEK and partially dependent on PB-kinase. In contrast, signaling in response to the phorbol ester, TPA, was partially dependent on PI3K but totally dependent on MEK-ERK. This indicates that the signal converges on ERK, suggesting the involvement of a PB-kinase upstream of a dominant MEK-ERK pathway. The differences found here between mouse and human insulin mitogenic signaling pathways indicate that there may be species differences between osteoblast signaling pathways, with mouse cells being independent and human cells being dependent on MEK for DNA synthesis in response to insulin. The effects of glucocorticoids on insulin mitogenic signaling in osteoblasts were also investigated, because chronic long-term steroid use results in excessive bone loss. The PTP inhibitor, sodium orthovanadate, reversed GC-impaired TPA- and FCS- induced proliferation in MBA-1SA and MG-63 preosteoblasts. PTPs, such as SHP-l and PTP-IB, dephosphorylate and inactivate phosphorylated kinases. Both SHP-l and PTPlB associated with kinases in the mitogenic signaling cascade of MBA-lS.4 preosteoblasts growing rapidly in 10% FCS. Further, SHP-I co-irnmunoprecipitated with active, tyrosine phosphorylated ERK, which may indicate that it can dephosphorylate and inactivate ERK. However, since the MEK-ERK or PB-kinase pathways are not important in insulin-induced proliferation in mouse osteoblasts, the PTPs are unlikely to be role players in the negative regulation of this signaling pathway. This was confirmed by the finding that vanadate was unable to reverse GC-induced decreases in insulinstimulated DNA synthesis. This suggests that vanadate-sensitive PTPs may not be important in the negative regulation of insulin-induced mouse osteoblast proliferation, and provides further evidence of a novel insulin mitogenic pathway in the MBA-lSA but not MG-63 osteoblastic cell line. / AFRIKAANSE OPSOMMING: Insulien is 'n mitogeen vir baie selle en gelei na binding aan die insulien reseptor, intrasellulêre seine via die klassieke, mitogeniese Raf-MEK-ERK of die metaboliese PB-kinase seintransduksie pad. 'n Insulien gebrek of tipe I diabetes veroorsaak osteopenie. Vetsugtige pasiënte met insulien weestandigheid of tipe II diabetes, 'n siekte wat geassosieer word met foutiewe insulien seintransduksie en hoë vlakke van sirkuierende insulien, het verhoogde of normale been mineraal digtheid (BMD). Die vraag of hiper insulin ernie 'n verlies aan beenmassa teëwerk word dikwels gevra. Teenstrydigheid oor die rol van insulien as 'n osteo-anaboliese stof bestaan egter steeds en hierdie vraag bly dus onbeantwoord. Dat insulien seintransduksie wel 'n kritiese rol speel in beenvormende osteoblaste is onlangs bevestig in studies met muise waarvan die geen vir IRS-l uitgeslaan is. Hierdie muise ontwikkel 'n lae omset osteopenie weens verswakte proliferasie en differensiasie. fn hierdie studie is gevind dat insulien wel in vitro as 'n osteoblast mitogeen kan funksioneer. Dit is in drie relatief onvolwasse (MBA-15.4, -15.6 muis en MG-63 mens) sellyne geillistreer, deur betekenisvolle verhogings in insulien-geaktiveerde proliferasie. In MBA-15.4 preosteoblaste is die persentasie verhoging in insulien-gestimuleerde proliferasie vergelykbaar met dié van die bekende mitogeniese forbolester, TPA. Die UMR-I06 sellyn het kenmerke van gedifferensieerde osteoblaste, en was baie minder responsief op insulien behandeling. Die verskil in die proliferasie vermoë van die verskillende sellyne kan die gevolg wees van verskille wat bestaan tussen spontaan getransformeerde sellyne of die stadium van sel differensiasie. 'n MEK 1/2 inhibitor, UO126 en 'n PB-kinase inhibitor, wortmannin, is gebruik om die insulien seintransduksie pad noodsaaklik vir die aktivering van ERK en osteoblast proliferasie te bepaal. In MBA-1S.4 muis pre-osteoblaste, was fetale kalf SenlTI1(FKS)-geinduseerde DNA sintese totaal afhanklik van MEK. Beide die MBA-15.4 en die meer volwasse MBA-15.6 muis osteoblaste was weerstandig teen die inhibitors op hulle eie, of in kombinasie. Verhoogde MEK-inhibitor konsentrasies het geen verdere effek gehad nie en in verskeie eksperimente is 'n verhoging in preliferasie selfs waargeneem met MEK-inhibisie. Hierdie resultate dui aan dat die klassieke insulien mitogeniese pad nie betrokke is in MBA-I5.4 gestimuleerde selproliferasie nie. Wortmannin het geen effek gehad op insulien- of20% FKS-gestimuleerde DNA sintese nie, maar het wel die aktivering van PB-kinase se metaboliese teiken, AktJPKB geinhibeer. Insulien seintransduksie aktiveer dus ERK deur beide MEK en PB-kinase, maar het geen effek op proliferasie gehad nie. FKS-gestimuleerde ERK aktivering en proliferasie was totaal afhanlik van MEK-ERK aktivering. Insulien-geaktiveerde DNA sintese in die mens MG-63 osteoblaste was gedeeltelik afhanklik van beide MEK en PB-kinase. Alhoewel IPA ook PB-kinase kon aktiveer, was dit totaal afhanklik van MEK vir DNA sintese. Dit dui aan dat daar 'n PB-kinase stroom-op van 'n dominante MEK-ERK seintransduksie pad voorkom. Die verskille wat ons dus waargeneem het in insulien mitogeniese seintransduksie tussen muis en mens, kan aandui dat insuliengestimuleerde seintranduksie paaie kan verskil van spesie tot spesie. Dit is bevestig met die muisselle wat onafhanklik is en mens selle wat afhanklik is van MEK aktivering vir insuliengeaktiveerde DNA sintese. Kroniese, langtermyn steroied behandeling kan beenverlies veroorsaak en die effek van glukokortikoide (GK) op die insulien mitogeniese pad in osteoblaste is dus ook ondersoek. Natrium-ortovanadaat, 'n proteien tirosien fosfatase (PIP) inhibitor het GK-verlaagde proliferasie in repons tot beide IPA- en FKS behandeling herstel in MBA-lSA en MG-63 preosteoblaste. PIPs soos SHP-l en PIP-l B funksioneer deur gefosforileerde kinases te defosforileer en dus te inaktiveer. Beide SHP-l and PIP-lB kon assosieer met kinases in die mitogeniese insulien seintransduksie pad van vinnig groeiende MBA-IS A preosteoblaste in 10% FKS. Verder het SHP-I ook geko-immunopresipiteer met aktiewe, tirosien-gefosforileerde ERK, wat aandui dat SHP-I met ERK assosieer om dit te defosforileer en inaktiveer. Die MEKERK of PB-kinase paaie is nie belangrik vir insulien-geaktiveerde seintransduksie in muis osteoblaste nie. Dit is dus onwaarskynlik dat die PIPs 'n rol sal speel in die negatiewe regulering van hierdie seintransduksie paaie. Die ontdekking dat vanadaat nie glukokortikoiedverlaagde insulien-geaktiveerde DNA sintese kan herstel nie, toon dat vanadaat-sensitiewe PIPs nie 'n rol speel in insulien-geaktiveerde proliferasie in muisselle nie. Hierdie bevinding het verder bevestig dat 'n nuwe insulien mitogeniese pad in die MBA-ISA, maar nie die MG-63 selle moontlik bestaan.

Meng, Fanbiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。 / 方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。 / 結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。 / 結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。 / Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied. / Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis. / Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality. / Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Liangliang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 128-144). / Abstract also in Chinese. / Declaration --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Chapter 1 --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Mesenchymal stem cells --- p.2 / Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2 / Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4 / Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5 / Chapter 1.2 --- Adipogenesis of MSCs --- p.6 / Chapter 1.3 --- Chondrogenesis of MSCs --- p.7 / Chapter 1.4 --- Osteogenesis of MSCs --- p.8 / Chapter 1.4.1 --- Regulators of osteogenesis --- p.9 / Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11 / Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13 / Chapter 1.5.1 --- ERK signaling pathway --- p.14 / Chapter 1.5.2 --- Wnt signaling pathway --- p.15 / Chapter 1.5.3 --- BMP signaling pathway --- p.17 / Chapter 1.6 --- Migration of MSCs --- p.20 / Chapter 1.7 --- Fracture healing --- p.22 / Chapter 1.8 --- Clinical application of MSCs --- p.23 / Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24 / Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25 / Chapter 1.9 --- Scope of the present study --- p.26 / Chapter 1.9.1 --- CRBP1 --- p.26 / Chapter 1.9.2 --- N-cadherin --- p.27 / Chapter 1.9.3 --- SOX11 --- p.27 / Chapter 1.10 --- Experimental scheme --- p.29 / Chapter 2 --- p.31 / Comparison between PB-MSCs and BM-MSCs --- p.31 / Chapter 2.1 --- Chapter introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.33 / Chapter 2.2.1 --- Cell culture --- p.33 / Chapter 2.2.2 --- Flow cytometry --- p.33 / Chapter 2.2.3 --- Adipogenic differentiation --- p.34 / Chapter 2.2.4 --- Osteogenic differentiation --- p.34 / Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35 / Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36 / Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38 / Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39 / Chapter 2.4 --- Discussion and future work --- p.40 / Chapter 3 --- p.41 / Role of CRBP1 in Differentiation and Migration of MSCs --- p.41 / Chapter 3.1 --- Chapter introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.46 / Chapter 3.2.1 --- Chemicals --- p.46 / Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46 / Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47 / Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48 / Chapter 3.2.5 --- Osteogenic differentiation --- p.50 / Chapter 3.2.6 --- Adipogenic differentiation --- p.50 / Chapter 3.2.7 --- Western blot --- p.51 / Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52 / Chapter 3.2.9 --- Cell migration assay --- p.52 / Chapter 3.2.10 --- Ectopic bone formation assay --- p.52 / Chapter 3.2.11 --- Statistical analysis --- p.53 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53 / Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56 / Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58 / Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61 / Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63 / Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64 / Chapter 3.4 --- Discussion --- p.66 / Chapter 3.5 --- Future work --- p.73 / Chapter 4 --- p.74 / Role of N-cadherin in Differentiation and Migration of MSCs --- p.74 / Chapter 4.1 --- Chapter introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.78 / Chapter 4.2.1 --- Chemicals --- p.78 / Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78 / Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79 / Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81 / Chapter 4.2.5 --- Western blot --- p.81 / Chapter 4.2.6 --- Ectopic bone formation assay --- p.82 / Chapter 4.2.7 --- Statistical analysis --- p.82 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83 / Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84 / Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86 / Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87 / Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89 / Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89 / Chapter 4.4 --- Discussion --- p.91 / Chapter 4.5 --- Future work --- p.94 / Chapter 5 --- p.96 / Role of SOX11 in Differentiation and Migration of MSCs --- p.96 / Chapter 5.1 --- Chapter introduction --- p.97 / Chapter 5.2 --- Materials and methods --- p.105 / Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105 / Chapter 5.2.2 --- Cell culture --- p.106 / Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106 / Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106 / Chapter 5.2.5 --- Adipogenic differentiation --- p.107 / Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107 / Chapter 5.2.6 --- Western blot --- p.108 / Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108 / Chapter 5.2.8 --- Cell migration --- p.110 / Chapter 5.2.9 --- Ectopic bone formation --- p.110 / Chapter 5.2.10 --- Fracture healing model and analysis --- p.111 / Chapter 5.2.11 --- Statistical Analysis --- p.112 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112 / Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113 / Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114 / Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115 / Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117 / Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118 / Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122 / Chapter 5.4 --- Discussion --- p.126 / Chapter 5.5 --- Future work --- p.131 / Appendix --- p.153

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.

Discordance in bone mass between young adult swimmers and soccer players may be a direct result of differences in bone loading patterns that influence bone mineralization during growth. Our aim was to evaluate whether sports participation (soccer and swimming) had an independent effect on bone mass accrual at the hip and lumbar spine in adolescent female athletes. We recruited boys and girls 10 to 14-years of age from Corvallis, Albany, Sweet Home, Salem, Eugene, and the greater Portland area. Bone mineral content (BMC, g) and bone mineral density (BMD, g/cm²) of the proximal left hip, spine, and whole body were assessed by dual energy x-ray absorptiometry (Hologic QDR 4500A; Hologic Inc., Waltham, MA, USA). We used ANCOVA and report that baseline BMC and BMD values of girl soccer players at the greater trochanter were significantly higher compared to controls and the swim group, and femoral neck BMC was significantly greater than the swimmers. At baseline, all boy groups were similar at the hip and spine. After 12-months, ANCOVA was also used to assess absolute change for BMC and BMD at the hip and spine. The girl soccer players had significantly more BMC and BMD at the greater trochanter as well as total hip BMD and lumbar spine BMC compared to the swimmers, but not the controls. The girl control group showed a significantly greater 12-month change for femoral neck and greater trochanter BMC than swimmers. Overall, the girl swimmers demonstrated a lower accumulation of bone mass during the 12-month study period. As for the boys, soccer players had a significantly higher 12-month change for femoral neck BMC than swimmers, but were similar at the spine. There were no differences between the boy control subjects and the swimmers for 12-month change values at the hip and spine. While preliminary and limited by the small sample size, our results indicate that after controlling for growth, soccer players gained significantly more BMC at the femoral neck than swimmers. Furthermore, exposing the young skeleton to impact loading exercise has site-specific benefits at the hip whereas prolonged training in a non-weight bearing environment may compromise skeletal acquisition. / Graduation date: 2004

It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.

Intrauterine growth restriction (IUGR) reduces bone mass by 10-30% and impairs arachidonic (AA) and docosahexaenoic (DHA) acid status in infants. Because AA and DHA enhance neonatal bone mass, the aim of this study was to determine the effects of dietary 0.5% AA and 0.2% DHA (w/w) prior to weaning on bone and growth. 40 guinea pigs were randomized to either a control (C) or low-protein diet (LP) during pregnancy and the C diet or the C diet with AA+DHA during lactation. Measurements included bone mass, metabolism, and strength, and erythrocyte lipid of sows and offspring from birth to 16 wk post-partum. The LP diet induced IUGR, while the AA+DHA increased bone mass by 5-20% in sows and offspring and corrected growth and bone mass in IUGR pups. Thus, AA+DHA provided in lactation rescues the growth trajectory in an IUGR state and is beneficial to maternal and neonatal bone mass.

To determine the influence of running on certain blood-born parameters that are involved in bone metabolism, serum levels of calcium, alkaline phosphatase (ALP, a marker of bone formation), growth hormone (hGH), and parathyroid hormone (PTH), were analyzed in 10 male subjects following a 40 min. run at 70% VO2max. Each trial was preceeded by 1 day of inactivity, a 8-12 hr. fast, and drawing of a baseline blood sample by venipuncture. All other blood samples were taken via an indwelling catheter which was inserted in an antecubital vein immediately following the completion of the exercise bout. When the catheter was in place, an "immediate post" sample was drawn. Subsequent samples were taken at 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, and 60 min. after the immediate post sample. Analysis of serum calcium concentrations demonstrated that levels were significantly elevated by 12% following exercise, going from a fasted level of 9.7 ± .53 mg/dl to post-exercise levels of 11.8 ± .73 mg/dl. Serum calcium remained elevated during the first 4 min. following exercise. By 5 min. post-exercise, calcium levels dropped to levels that were significantly lower than the post-exercise sample. However, serum alkaline phosphatase did not change significantly following exercise, as the values remained within normal range throughout the experimental period. Concentrations tended to decrease over time but were not significantly lower than the preor post-exercise levels by the end of the sampling period. Serum concentrations of hGH were more than doubled following a single bout of exercise, going from 4.0 ± 0.98 ng/ml before exercise to 8.8 ± 1.6 ng/ml immediately post-exercise. Following this initial rise, hGH progressively declined and returned to baseline values by 30 min. post-exercise. The concentrations of PTH did not change significantly following exercise. The postexercise sample tended to be higher than baseline values but were not significantly different. The results presented here indicate that an exercise bout 40 min. at 70% V02max results in an elevation of serum calcium and hGH, but does not alter PTH secretion or ALP activity. The data presented in this study indicate that the temporary rise in calcium following exercise is unrelated to PTH. It is hypothesized that the increase in calcium that we observed is attributable to lactate accumulation that would result from an exercise bout of this nature. The buildup of lactic acid and drop in pH causes a dissolution of the crystaline calcium hydroxyapatite compartment of the skeleton, thus causing an increase in ionized calcium. It is not known whether a single bout of exercise can influence hormonal secretion to a sufficient degree to affect bone density, but the hormonal changes demonstrated here could be involved in long-term effects of training. / School of Physical Education

Since vitamin D deficiency is common at birth, the objective of this study was to test if postnatal vitamin D supplementation would normalize bone mineralization. Forty guinea pigs were randomized to receive a diet with or without vitamin D3 during pregnancy. Newborn pups were randomized to receive 10 IU of vitamin D3 or a placebo daily until d28. Measurements at birth and d28 included whole body and regional bone mass, osteocalcin and deoxypyridinoline, plus biomechanical testing of excised tibias and femurs. Offspring from deficient sows had lower body weight, whole body and tibia bone mineral content (BMC) and lower osteocalcin and biomechanical integrity. By d28 this group had lower whole body bone density and femur BMC, unless supplemented. Interactions with gender showed males continued to have low 25(OH)D despite supplementation. Therefore, neonates born to sows with dietary vitamin D deficiency require supplemental vitamin D to support normal bone mineral accretion.