This thesis is based on studies of the ten-year community-based intervention programme entitled, the Vadstena Osteoporosis Prevention Project (VOPP). The specific aims of the research were to describe the effects of physical activity and training programmes on bone mass and balance performance in adults, to determine whether a fall risk prevention programme could motivate personal actions among the elderly, to ascertain whether the intervention programme could reduce the incidence of forearm and hip fractures. Two studies addressed training programmes for middle-aged and old people. First, VOPP participants who were aged 40–70 years and had low forearm bone mineral density (BMD) values were invited to take part in a one-year weight-bearing training study. Thirty of those individuals were included in the investigation. Additional bone mass measurements were performed at the hip and the lumbar spine, and balance and aerobic capacity were also tested. The training programme was performed twice a week (I). In the second study, healthy persons aged 70–75 years were invited to participate in a balance-training study. Fifteen persons joined an exercise group, and another fifteen were controls. The training programme comprised specific balance exercises and was carried out twice a week for nine weeks (II). The association between forearm BMD values and several lifestyle factors was explored in random samples of the population aged 20–72 years (n=880) in a cross-sectional study (III). Another study explored the association between calcaneal stiffness, forearm BMD, and lifestyle factors amongst participants aged 20–79 years (n=956) at the final registration of the VOPP (V). Effects of the VOPP interventions directed at environmental risk factors for falls and the promotion of physical activity were examined in people aged ≥ 65 years (IV). The incidence of forearm and hip fractures was studied amongst middle-aged and elderly individuals in the intervention and the control communities during the study period 1987–2001 (VI). The exercise group (n=15) in the weight-bearing training study showed increases in BMD at the greater trochanter (p<0.01), one-leg stance balance with the eyes closed and coordination tests (p<0.05), and aerobic capacity (p<0.05). No significant difference was found when the groups were compared concerning changes in the different tests during the intervention period (I). In the balance-training study, the exercise group showed post-training improvement in the following tests: standing on the right leg with eyes closed (p<0.01), standing on the right leg (p<0.01) and on the left leg (p<0.05) while turning the head, and walking 30 metres (p<0.01). There were significant differences between the groups in these tests when changes were compared at the post-intervention test (II). Age (p<0.0001) and body mass index (p≤.0001) were associated with forearm BMD in both sexes. Reported moderate physical activity levels in men were positively associated with forearm BMD (p<0.05) (III). In both sexes, reported moderate (p<0.05) and high (women p<0.05 and men p<0.001) physical activity levels were positively associated with calcaneal stiffness. The correlation coefficient between forearm BMD and calcaneal stiffness was 0.58 in women and 0.34 in men (V). Persons aged ≥ 65 years at the follow-up in 1994 reported more use of shoe/cane spikes and moderate physical activity levels compared to controls (IV). There was no change in the general incidence of forearm and hip fractures between the communities for the study period. However, there was a tendency towards decreasing incidence of forearm and trochanteric hip fracture in both sexes during the late intervention period in the intervention community (VI). A community-based intervention programme aimed at reducing the incidence of osteoporotic fractures must be regarded as a long-term project and should preferably be monitored over an extended post-intervention period. / On the day of the public defence the statuses of articles IV and V were Submitted and VI was Manuscript

Wong Yin-Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 119-135). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Publications --- p.v / Acknowledgements --- p.vi / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xiv / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Osteoporosis --- p.1 / Chapter 1.1.1 --- Consensus statement --- p.1 / Chapter 1.1.2 --- Epidemiology and outcomes --- p.4 / Chapter 1.1.2.1 --- Hip fractures --- p.4 / Chapter 1.1.2.2 --- Vertebral fractures --- p.5 / Chapter 1.1.2.3 --- Wrist fractures --- p.7 / Chapter 1.1.3 --- Postmenopausal osteoporosis --- p.8 / Chapter 1.1.3.1 --- Pathogenesis --- p.8 / Chapter 1.1.3.1.1 --- Genetics --- p.11 / Chapter 1.1.3.1.2 --- Bone remodeling --- p.14 / Chapter 1.1.3.1.3 --- Calcium homeostasis --- p.21 / Chapter 1.1.3.1.4 --- Life style 一 nutrition and exercise --- p.26 / Chapter 1.1.3.2 --- Current pharmacological treatment --- p.27 / Chapter 1.1.3.2.1 --- Introduction --- p.27 / Chapter 1.1.3.2.2 --- Limitations --- p.31 / Chapter 1.2 --- Traditional Chinese medicine --- p.33 / Chapter 1.2.1 --- The Kidney --- p.33 / Chapter 1.2.2 --- Kidney-tonifying herbs --- p.33 / Chapter 1.3 --- Aim of the studies --- p.36 / Chapter Chapter 2. --- Materials and methods --- p.38 / Chapter 2.1 --- Kidney-tonifying herbs and herbal formula --- p.38 / Chapter 2.1.1 --- Sources --- p.38 / Chapter 2.1.2 --- Herbal extract preparation --- p.38 / Chapter 2.2 --- Animal study --- p.40 / Chapter 2.2.1 --- Reagents --- p.40 / Chapter 2.2.2 --- Animal care --- p.40 / Chapter 2.2.3 --- Herbs and herbal formula preparations for animal studies --- p.41 / Chapter 2.2.4 --- Experimental design --- p.41 / Chapter 2.2.5 --- Gene expression study --- p.44 / Chapter 2.2.5.1 --- Tissue preparation --- p.44 / Chapter 2.2.5.2 --- Isolation of total RNA --- p.45 / Chapter 2.2.5.3 --- Complementary DNA synthesis --- p.47 / Chapter 2.2.5.4 --- Real-time polymerase chain reaction analysis --- p.47 / Chapter 2.3 --- Cell culture study --- p.49 / Chapter 2.3.1 --- Reagents --- p.49 / Chapter 2.3.2 --- Cell lines --- p.49 / Chapter 2.3.2.1 --- "Rat osteosarcoma cell line, UMR-106" --- p.49 / Chapter 2.3.2.2 --- "Human breast cancer cell line, MCF-7" --- p.50 / Chapter 2.3.2.3 --- Cell culture techniques --- p.50 / Chapter 2.3.3 --- Herbs preparations for cell culture --- p.51 / Chapter 2.3.4 --- Cell viability assay --- p.51 / Chapter 2.3.5 --- Cellular alkaline phosphatase activity assay --- p.52 / Chapter 2.3.6 --- Matrix mineralization assay --- p.54 / Chapter 2.3.7 --- Competitive estrogen receptor binding assay --- p.56 / Chapter 2.4 --- Statistical analyses --- p.58 / Chapter Chapter 3. --- Results --- p.59 / Chapter 3.1 --- Extraction yields of Kidney-tonifying herbs and herbal formula --- p.59 / Chapter 3.2 --- Effects of Kidney-tonifying herbs and herbal formula on the gene expressions of calcium absorption and reabsorption related genes --- p.61 / Chapter 3.2.1 --- Gene expression of 25-hydroxyvitamin D3-1 alpha-hydroxylasein the kidney --- p.62 / Chapter 3.2.2 --- Gene expression of vitamin D receptor in the duodenum --- p.65 / Chapter 3.2.3 --- Gene expression of calbindin D9K in the duodenum --- p.67 / Chapter 3.2.4 --- Gene expression of vitamin D receptor in the kidney --- p.69 / Chapter 3.2.5 --- Gene expression of calbindin D28K in the kidney --- p.71 / Chapter 3.3 --- Effects of Kidney-tonifying herbs on osteoblastic UMR-106 cell line --- p.73 / Chapter 3.3.1 --- Effects of Kidney-tonifying herbs on the cell viability of UMR-106 cells --- p.73 / Chapter 3.3.2 --- Effects of Kidney-tonifying herbs on the osteoblastic differentiation of UMR-106 cells --- p.76 / Chapter 3.3.2.1 --- Cellular alkaline phosphatase activity --- p.76 / Chapter 3.3.2.2 --- Degree of matrix mineralization --- p.80 / Chapter 3.4 --- Estrogen receptor binding activities of Kidney-tonifying herbs --- p.85 / Chapter Chapter 4. --- Discussion --- p.89 / Chapter 4.1 --- Safety of Kidney-tonifying herbs and herbal formula --- p.89 / Chapter 4.2 --- Kidney-tonifying herbs and herbal formula preserve bone mineral density --- p.93 / Chapter 4.3 --- Kidney-tonifying herbs and herbal formula modulate calcium homeostasis --- p.97 / Chapter 4.3.1 --- "Roles in renal synthesis of the hormonally active form of vitamin D: 1,25-dihydroxyvitamin D3" --- p.97 / Chapter 4.3.2 --- Roles in calcium absorption in the duodenum --- p.99 / Chapter 4.3.3 --- Roles in calcium reabsorption in the kidney --- p.102 / Chapter 4.3.4 --- Summary --- p.104 / Chapter 4.4 --- Kidney-tonifying herbs modulate bone formation --- p.106 / Chapter 4.4.1 --- Effects on osteoblast proliferation --- p.106 / Chapter 4.4.2 --- Effects on osteoblastic differentiation --- p.107 / Chapter 4.4.3 --- Summary --- p.108 / Chapter 4.5 --- Kidney-tonifying herbs interact with estrogen receptor --- p.110 / Chapter 4.6 --- Active ingredients of Kidney-tonifying herbs --- p.111 / Chapter 4.7 --- Limitations of the present studies --- p.115 / Chapter 4.8 --- Conclusion and future prospect --- p.117 / References --- p.119

傳統中醫中藥理論遵從"腎主骨"概念。因此，中醫在治療與骨有關的疾病時一般都處方"補腎"類中藥。 / ELP是一例中藥草本 "補腎" 複方。其包含三種中藥，包括淫羊藿（E）、女貞子（L）和補骨脂（P）。動物體內實驗和臨床研究已證明ELP有效治療絶經後骨質疏鬆症。可是，經口服吸收後的血清中的ELP有效物質對細胞的成骨影響從未進行過相關研究。ELP對預防在缺乏體力活動下所引起的骨質疏鬆症的療效也屬未知。此外，基於其"補腎"的特性，ELP可能潛在著能促進骨折癒合的功能。本研究的目的包括研究血清中ELP的有效物質在細胞和分子水平上的護骨能力，並測試其對預防於失重狀態下引起的骨質疏鬆症（慢性骨紊亂）的效能。本研究還旨在考察 ELP在促進骨癒合 （急性骨紊亂）上的作用。本研究分為三部分。 / 第一部分 -- 骨代謝的體外研究:健康大鼠分別口服草本配方ELP、EL、及單味中草藥提取物E或L、並以蒸餾水作為對照（H2O），口服給藥二小時後收集其血清作體外血清藥理學研究。分別考察含藥血清對各細胞系包括UMR106、RAW264.7、和從大鼠骨中分離出的骨髓間充質幹細胞（MSC）的增殖和分化屬性的影響，並以液質聯用技術（LC-MS）來分析血清內所含中藥的化學成份。 / 第二部分 -- 骨質疏鬆症的體內研究:以尾吊雄性大鼠作為卸荷狀態骨質疏鬆症的動物模型。在不同的給藥組中，大鼠口服高中低三種劑量的ELP（ELP-H、ELP-M和ELP-L），或三個不同抗骨質疏鬆藥物，包括雷洛昔芬（Ral），阿侖膦酸鈉（Aln）和雷奈酸鍶（Strn）作為陽性對照組，並以蒸餾水為安慰劑對照（TS）。另一組大鼠則沒有尾吊，作為正常對照（Non-TS）。本部分分析在吊尾期間大鼠體內生化指標和骨密度（BMD）的變化，及其後各組在骨小梁微結構和骨骼生物力學上的差異。 / 第三部分 -- 骨缺損癒合的體內研究:兩個鑽孔性骨缺損模型分別建立於老年雌性大鼠的左股骨骨幹和右脛骨近端骺端。其後動物分成4組：（1）ELP 口服給藥（ELP）；（2）CDNR外敷治療（CDNR為另一中藥複方，包含紅花（C）、續斷（D）、三七（N）和大黃（R））；（3）ELP口服給藥結合CDNR外敷治療（ELP+CDNR）；（4）和蒸餾水餵養（Control）。通過監測骨缺損癒合的過程、檢測大鼠血液中生化標誌物的變化、骨骼生物力學測試和形態計量學分析，考察ELP及其與CDNR在骨缺損癒合上的協同作用。 / 第一部分的結果顯示，口服給藥二小時後，大鼠血清中淫羊藿的標記化合物淫羊藿苷（icariin）無被檢出。在EL或E的給藥大鼠血清中，檢出淫羊藿苷的其中一個代謝產物icariside I；而其另一個代謝產物icariside II，則在ELP的給藥大鼠血清中檢測到。L和P的常見標記化合物則能從相應餵飼L和P的大鼠血清中檢出。體外血清藥理學研究結果表明含藥（ELP）大鼠血清對細胞無毒性作用，且能促進 UMR106 細胞增殖和上調其Runx2 基因表達。然而，含藥血清無增加UMR106細胞的鹼性磷酸酶活性和鈣沉積。它抑制 RAW264.7細胞的分化及其基質金屬蛋白酶9（MMP-9）和組織蛋白酶 K的基因表達。它亦能促進MSC細胞的增殖，增強其鹼性磷酸酶活性和Runx2與ALP基因的表達。 / 第二部分的結果指出ELP-H能減少吊尾大鼠股骨遠端及腰椎骨密度的百分比損失，抵抗股骨遠端骨小梁微結構惡化和加強股骨骨幹骨缺損部位的生物力學特性。此外，ELP-H還能降低血液骨鈣素和抗酒石酸酸性磷酸酶5b（TRAP5b）的濃度。研究亦發現ELP對骨密度、結構參數和生化指標的影響存在劑量依賴性。整體上而言，ELP在預防卸荷骨質疏鬆症的影響類似於Ral和Aln，而非Strn。 / 第三部分的結果表明，從顯微電腦掃描或形態計量學上分析，所有實驗組跟對照組間均沒有顯著性差異。但值得注意的是，ELP+CDNR大大提高了股骨骨幹骨缺損在癒合過程中的歸一化生物力學屬性。而ELP單獨用藥則減少了TRAP5b的濃度。 / 總之，這項研究結論出血清藥理學研究加上LC-MS的應用能作為找出中藥中有效成分的有效途徑。本研究還展示ELP的含藥血清對骨細胞有護骨作用。ELP可防預在卸荷狀態下形成的骨質疏鬆症，它還有助於提升外敷中藥複方CDNR在骨缺損癒合過程中的療效。從這項研究的三個部分中歸納出的共同點說明，儘管ELP擁有刺激成骨的能力，它的護骨作用主要是透過它的抗骨吸收效果。ELP在慢性（防止骨質疏鬆症）和急性（促進骨癒合）骨紊亂上均有療效。 / Traditional Chinese Medicine (TCM) claims that bone health lies in the functioning of the "Kidneys". When the "Kidney" is strong, our body can stimulate growth and transformation of the bone marrow, which nourishes and strengthens the skeleton. Therefore, "Kindey-tonifying" herbs are usually used to cure bone diseases. / ELP is a "Kidney-tonifying" Chinese herbal formula containing three Chinese herbs including Herba Epimedii (E), Fructus Ligustri Lucidi (L) and Fructus Psoraleae (P). It has been proven effective to treat postmenopausal osteoporosis through in vivo and clinical studies. However, ELP is for oral administration. The osteogenic properties of its post-absorption metabolites have never been studied. The efficacy of ELP on prevention of osteoporosis development due to physical inactivity is also unknown. With its "Kindey-tonifying" property, ELP is also considered as a potential agent to facilitate fracture healing. / The aims of this study included to investigate the osteoprotective effects of ELP metabolites at cellular and molecular levels and to prove the efficacy of ELP on prevention of osteoporosis development in unloading condition - a chronic bone disorder. It also aimed to study the effect of ELP on promotion of bone defect healing - an acute bone disorder. This study was divided into three parts. / Part 1 - in vitro study of bone metabolism: Healthy rats were fed with herbal formula ELP or EL, single herbal extracts of E or L or distilled water as control (H₂O). Sera were then collected for in vitro seropharmacological study. Cell lines including UMR106 and RAW264.7, as well as mesenchymal stem cell (MSC) isolated from rats, were cultured with the sera. Their proliferation and differentiation properties of the cells were analyzed. In addition, the chemical profiles of the herbal extracts within the sera were analyzed using liquid chromatography-mass spectrometry (LC-MS). / Part 2 - in vivo study of osteoporosis: Tail-suspension male rats were used as the unloading osteoporotic animal model. The rats in different groups were fed with three different doses of ELP (ELP-H, ELP-M and ELP-L), or three different anti-osteoporosis drugs including raloxifene (Ral), alendronate (Aln) and strontium ranelate (Strn) as positive controls or distilled water as placebo control (TS). One group of rats was non-tail-suspended as normal control (Non-TS). Changes in bone mineral density (BMD), microarchitecture of trabeculae and biomechanical properties of the bone of the rats were analyzed. Changes in biochemical markers within the tail-suspension period were also studied. / Part 3 - in vivo study of bone defect healing: two drilled-hole bone defects were created in the diaphysis of left femur and proximal metaphysis of right tibia, respectively, of aged female rats. Animals were divided into 4 groups: (1) administered with ELP orally (ELP); (2) treated with another herbal formula CDNR containing Carthami Flos (C), Dipsaci Radix (D), Notoginseng Rhizoma (N) and Rhei Rhizoma (R) topically (CDNR); (3) treated with oral ELP and topical CDNR at the same time (ELP+CDNR); and (4) fed with distilled water (Control). The effects of ELP and the synergistic effects of ELP+CDNR on facilitation of the bone defect healing were monitored in vivo using viva-CT and through measurement of biochemical markers biweekly. After euthanasia of the rats, the bones were harvested for biomechanical test and histomorphometrical analysis. / Results: Part 1 revealed that the common marker compound, icariin, had not been detected in the sera of all the rats. Instead, one of the metabolites of E, icariside I, was found in the sera of the rats fed with EL or E, while another metabolite, icariside II, was detected in the serum of the rats fed with ELP. Common marker compounds of L and P were observed in the sera of the rats fed with the herbal items accordingly. The in vitro studies in this Part showed that there was no cytotoxic effect of the rat sera on the cells. The post-absorbed ELP metabolites in rat serum promoted UMR106 proliferation by 25.7%, (p < 0.05) and upregulated the Runx2 gene expression by 1.18 fold (p < 0.05) after cultured for 2 and 3 days, respectively. However, they could not increase the ALP activity and calcium deposition of UMR106. They also inhibited RAW264.7 differentiation by 29.2 % (p < 0.05) and downregulated the MMP9 and Cathepsin K gene expression of RAW264.7 by 0.46 (p < 0.05) and 0.36 (p < 0.01) fold, respectively. The ELP metabolites promoted the proliferation of MSC by 14.4 % (p < 0.001) and resulted in 42.6 % higher ALP activity than the control serum (p < 0.05). They also upregulated the Runx2 and ALP gene expression at both Day 4 and Day 7 of culture significantly. / Part 2 showed that compared with the tail-suspension control (TS), ELP in high dose (ELP-H) reduced the percentage loss of total and trabecular BMD by 5.46 and 8.52 %, respectively (p < 0.05 both) in distal femur, and by 4.67 % (p < 0.05) in trabecular region of lumbar spine of the tail-suspended rats. Analysis from micro-CT showed that microarchitectural parameters BV/TV, Tb.Th and TV density of the distal femur of ELP-H were 17.62, 11.90 and 8.09 % higher than those of the TS (p < 0.05, for all). 3-point bending test on mid-shaft femur of the rats revealed that the yield load, ultimate load and stiffness of the drill-defect of ELP-H were higher than those of TS significantly. All of the biochemical markers decreased significantly from baseline (Day 0) to Day 28 in ELP-H. In addition, osteocalcin and TRAP5b concentrations of ELP-H were lower than those of TS significantly at Day 28. The effect of ELP on BMD, microarchitectural parameters and biochemical markers were in dose-dependent manner. In general, the osteoprotective effect of ELP-H on unloading bone was similar to Ral and Aln, but not Strn. / Part 3 indicated no significant difference in BV/TV and BMD among all groups at each time point. Histomorphometrical analysis from fluorescent labeling and Goldner’s trichrome staining showed no statistical difference in new bone formation between the Control and other treatment groups. Notably, the normalized yield load, ultimate load and failure of ELP+CDNR were significantly higher than those of Control by 20.38 % (p < 0.05), 23.17 % (p< 0.001) and 25.55 % (p< 0.001), respectively. Analysis on the change of biochemical markers showed that the bone formation marker BALP increased while bone resorption markers Dpd and TRAP5b decreased within the 42-day monitoring period. BALP activity of both Control and ELP increased significantly but only ELP reduced the TRAP5b concentrations starting from Day 14 post-op. There was no statistical difference when the concentrations of the biochemical markers were compared horizontally among the 4 groups at the same time point. / In conclusion, the current study demonstrated that seropharmacological study incorporating with the application of LC-MS can be a potential efficient approach to find out active ingredients of medicine herbs. Post-absorbed metabolites of ELP also showed their osteoprotective effects on bone cells. Aqueous extract of ELP could prevent the development of osteoporosis in unloading condition and such effect was dose-dependent. It also helped elevating the efficacy of a topical applied herbal formula CDNR on improving the bone strength of healing bone defects. A common finding from the 3 parts of this study illustrated that the osteoprotective effect of ELP was mainly achieved by its anti-resorptive efficacy on bone, although it possess an ability to stimulate osteoblastogenesis. ELP was found effective for both chronic (prevent osteoporosis development) and acute (facilitate bone healing) bone 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. / 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. / Siu, Wing Sum. / "November 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-227). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.vi / ACKNOWLEDGEMENTS --- p.ix / TABLE OF CONTENTS --- p.xi / LIST OF FIGURES --- p.xvii / LIST OF TABLES --- p.xxiii / PUBLICATIONS --- p.xxiv / ABBREVIATION --- p.xxv / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1.1 --- TRADITIONAL CHINESE MEDICINE (TCM) AND BONE DISEASES --- p.1 / Chapter 1.2 --- CELLULAR AND MOLECULAR MECHANISMS ON BONE METABOLISM --- p.2 / Chapter 1.2.1 --- Bone formation by osteoblast --- p.3 / Chapter 1.2.2 --- Bone resorption by osteoclasts --- p.4 / Chapter 1.3 --- OSTEOPOROSIS --- p.5 / Chapter 1.3.1 --- Postmenopausal osteoporosis --- p.6 / Chapter 1.3.2 --- Disuse osteoporosis --- p.8 / Chapter 1.3.3 --- Basic principle of TCM on osteoporosis --- p.10 / Chapter 1.3.4 --- Common Chinese herbal medicine reported to have anti-osteoporotic effects --- p.11 / Chapter 1.4 --- BONE FRACTURE --- p.11 / Chapter 1.4.1 --- Biology and repair of bone fracture --- p.12 / Chapter 1.4.2 --- TCM on promotion of fracture healing --- p.13 / Chapter 1.4.3 --- Theories of TCM on fracture healing --- p.15 / Chapter CHAPTER 2: --- OSTEOPOROSIS AND HERBS --- p.16 / Chapter 2.1 --- CHINESE HERBAL MEDICINE SELECTED IN THIS PART --- p.16 / Chapter 2.2 --- DESIGN OF STUDY --- p.19 / Chapter 2.3 --- HYPOTHESES AND OBJECTIVES --- p.19 / Chapter 2.4 --- BACKGROUND OF THE STUDY --- p.23 / Chapter 2.4.1 --- In vitro study of ELP on bone cells --- p.23 / Chapter 2.4.2 --- In vivo study of ELP on postmenopausal osteoporosis --- p.23 / Chapter 2.4.3 --- Clinical study of ELP on postmenopausal osteoporosis --- p.24 / Chapter CHAPTER 3: --- PART 1 IN VITRO SEROPHARMACOLOGICAL STUDY ON OSTEOPOROSIS --- p.26 / Chapter 3.1 --- OBJECTIVES --- p.26 / Chapter 3.2 --- SEROPHARMACOLOGICAL APPROACH TO STUDY ELP --- p.26 / Chapter 3.3 --- TYPES OF CELLS INVOLVED IN THE CURRENT STUDY --- p.27 / Chapter 3.3.1 --- UMR106 --- p.28 / Chapter 3.3.2 --- RAW264.7 --- p.28 / Chapter 3.3.3 --- Mesenchymal stem cell (MSC) --- p.28 / Chapter 3.4 --- IN VITRO ASSESSMENTS ON BONE METABOLISM --- p.29 / Chapter 3.4.1 --- Bone formation --- p.29 / Chapter 3.4.1.1 --- 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay --- p.29 / Chapter 3.4.1.2 --- Bromodeoxyuridine (BrdU) assay --- p.30 / Chapter 3.4.1.3 --- Total alkaline phosphatase (ALP) activity measurement --- p.30 / Chapter 3.4.1.4 --- Calcium deposition analysis --- p.30 / Chapter 3.4.2 --- Bone degradation --- p.31 / Chapter 3.4.2.1 --- Tartrate-resistant acid phosphatase (TRAP) staining --- p.31 / Chapter 3.4.3 --- Phenotypic markers of cells involved in bone remodeling using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) --- p.31 / Chapter 3.5 --- MATERIAL AND METHODS --- p.37 / Chapter 3.5.1 --- Preparation of herbal extracts --- p.37 / Chapter 3.5.2 --- Serum preparation for seropharmacological study --- p.38 / Chapter 3.5.2.1 --- Administration of herbal extracts and blood collection --- p.38 / Chapter 3.5.2.2 --- Serum preparation --- p.38 / Chapter 3.5.3 --- Analysis of marker compounds in serum using liquid chromatographymass spectrometry (LC-MS) --- p.39 / Chapter 3.5.3.1 --- Serum preparation --- p.39 / Chapter 3.5.3.2 --- Operation of LC-MS --- p.39 / Chapter 3.5.4 --- Isolation and characterization of MSC from bone marrow --- p.40 / Chapter 3.5.5 --- Cell culture --- p.42 / Chapter 3.5.5.1 --- General materials --- p.42 / Chapter 3.5.5.2 --- UMR106 --- p.43 / Chapter 3.5.5.3 --- RAW264.7 --- p.44 / Chapter 3.5.5.4 --- Bone Marrow MSC --- p.45 / Chapter 3.5.6 --- Assays analyzing the responses of cells on the effect of metabolites of herbs in serum --- p.46 / Chapter 3.5.6.1 --- General materials --- p.46 / Chapter 3.5.6.2 --- Assays for bone formation --- p.50 / Chapter 3.5.6.3 --- Assays for bone degradation --- p.55 / Chapter 3.5.7 --- Statistical analysis --- p.56 / Chapter 3.6 --- RESULTS --- p.57 / Chapter 3.6.1 --- Chemical characterization of ELP extract --- p.57 / Chapter 3.6.2 --- Marker compounds found in rat serum using LC-MS --- p.58 / Chapter 3.6.3 --- Effects of herbal metabolites on UMR106 --- p.61 / Chapter 3.6.3.1 --- Effect on cell viability --- p.61 / Chapter 3.6.3.2 --- Effects on cell proliferation and differentiation --- p.61 / Chapter 3.6.3.3 --- Regulation on osteogenesis through gene expression --- p.63 / Chapter 3.6.4 --- Effects of herbal metabolites on RAW264.7 --- p.67 / Chapter 3.6.4.1 --- Effect on cell viability --- p.67 / Chapter 3.6.4.2 --- Inhibitory effect on RAW264.7 --- p.67 / Chapter 3.6.4.3 --- Regulation on osteoclastogenesis through gene expression --- p.67 / Chapter 3.6.5 --- Effects of herbal metabolites on bone marrow mesenchyma stem cell (MSC) --- p.70 / Chapter 3.6.5.1 --- Confirmation of MSC isolated from bone marrow of rat using flow cytometry --- p.70 / Chapter 3.6.5.2 --- Effect on cell viability --- p.70 / Chapter 3.6.5.3 --- Effects on cell proliferation and differentiation --- p.71 / Chapter 3.6.5.4 --- Regulation on osteogenesis through gene expression --- p.71 / Chapter 3.7 --- DISCUSSION --- p.75 / Chapter CHAPTER 4: --- PART 2 IN VIVO STUDY ON DISUSE OSTEOPOROSIS . --- p.83 / Chapter 4.1 --- OBJECTIVES --- p.83 / Chapter 4.2 --- POTENTIAL EFFECT OF ELP ON DISUSE OSTEOPOROSIS --- p.83 / Chapter 4.3 --- ANIMAL MODELS FOR OSTEOPOROSIS STUDY --- p.84 / Chapter 4.3.1 --- Conventional ovariectomized animal model for the studies of osteoporosis --- p.85 / Chapter 4.3.2 --- Animal models for study of disuse osteoporosis --- p.85 / Chapter 4.3.2.1 --- Bandaging or casting --- p.86 / Chapter 4.3.2.2 --- Tail-suspension (TS) --- p.86 / Chapter 4.4 --- ASSESSMENTS ON DISUSE OSTEOPOROSIS DEVELOPMENT --- p.87 / Chapter 4.4.1 --- Bone mineral density (BMD) measurement --- p.87 / Chapter 4.4.2 --- Micro-architecture analysis --- p.87 / Chapter 4.4.3 --- Bone strength assessment --- p.88 / Chapter 4.4.4 --- Bone turnover monitoring by measuring biochemical markers --- p.89 / Chapter 4.4.4.1 --- Bone formation markers --- p.89 / Chapter 4.4.4.2 --- Bone resorption markers --- p.91 / Chapter 4.5 --- MATERIAL AND METHODS --- p.95 / Chapter 4.5.1 --- Preparation of herbal extracts --- p.95 / Chapter 4.5.2 --- Tail-suspension rat model --- p.95 / Chapter 4.5.3 --- Animal arrangement and grouping --- p.97 / Chapter 4.5.4 --- Administration of herbal extracts and drugs --- p.97 / Chapter 4.5.5 --- Assessments on disuse osteoporosis development --- p.98 / Chapter 4.5.5.1 --- Bone mineral density measurement using Peripheral Quantitative Computed Tomography (pQCT) --- p.98 / Chapter 4.5.5.2 --- Bone micro-architecture analysis using Micro-computed Tomography (μCT) --- p.99 / Chapter 4.5.5.3 --- Bone strength assessment through biomechanical bending test --- p.100 / Chapter 4.5.5.4 --- Bone turnover monitoring by measuring biochemical markers --- p.100 / Chapter 4.5.5.4.1 --- Serum collection --- p.100 / Chapter 4.5.5.4.2 --- Measurements of biochemical markers --- p.101 / Chapter 4.5.6 --- Statistical analysis --- p.105 / Chapter 4.6 --- RESULTS --- p.106 / Chapter 4.6.1 --- Effects of ELP on bone mineral density (BMD) --- p.106 / Chapter 4.6.2 --- Effects of ELP on bone micro-architecture --- p.118 / Chapter 4.6.3 --- Effects of ELP on biomechanics of bone --- p.122 / Chapter 4.6.4 --- Effects of ELP on bone turnover --- p.125 / Chapter 4.7 --- DISCUSSION --- p.132 / Chapter CHAPTER 5: --- PART 3 IN VIVO STUDY ON BONE DEFECT HEALING --- p.140 / Chapter 5.1 --- HERBAL ITEMS SELECTED IN THIS PART --- p.140 / Chapter 5.2 --- DESIGN OF STUDY --- p.143 / Chapter 5.3 --- HYPOTHESES AND OBJECTIVES --- p.144 / Chapter 5.4 --- SPECIFIC STRATEGY ON PROMOTION OF FRACTURE HEALING OF TCM --- p.144 / Chapter 5.5 --- POTENTIAL EFFECT OF ELP ON BONE HEALING --- p.144 / Chapter 5.6 --- ANIMAL MODELS --- p.146 / Chapter 5.6.1 --- Bone fracture model --- p.147 / Chapter 5.6.2 --- Drill-hole bone defect model --- p.147 / Chapter 5.7 --- ASSESSMENTS ON BONE HEALING --- p.149 / Chapter 5.7.1 --- Micro-architecture analysis --- p.149 / Chapter 5.7.2 --- Bone strength assessment --- p.150 / Chapter 5.7.3 --- Bone turnover monitoring by measuring biochemical markers --- p.151 / Chapter 5.7.4 --- Histomorphometry --- p.151 / Chapter 5.8 --- MATERIALS AND METHODS --- p.153 / Chapter 5.8.1 --- Preparation of herbal extracts --- p.153 / Chapter 5.8.1.1 --- ELP --- p.153 / Chapter 5.8.1.2 --- CDNR --- p.153 / Chapter 5.8.2 --- Production of drill-hole bone defect --- p.154 / Chapter 5.8.2.1 --- Femur --- p.155 / Chapter 5.8.2.2 --- Tibia --- p.155 / Chapter 5.8.2.3 --- Animal arrangement and grouping --- p.157 / Chapter 5.8.3 --- Herbal formulae administration and application --- p.157 / Chapter 5.8.3.1 --- Oral administration --- p.157 / Chapter 5.8.3.2 --- Topical application --- p.157 / Chapter 5.8.4 --- Assessments on bone healing --- p.158 / Chapter 5.8.4.1 --- Bone micro-architecture and bone density measurement using in vivo micro-computed tomography (vivaCT) --- p.158 / Chapter 5.8.4.2 --- Bone strength assessment through biomechanical bending test --- p.159 / Chapter 5.8.4.3 --- Bone turnover monitoring by measuring biochemical markers --- p.160 / Chapter 5.8.4.4 --- Histomorphometry --- p.160 / Chapter 5.8.4.4.1 --- Fluorochrome double labeling --- p.160 / Chapter 5.8.4.4.2 --- Tissue processing and sectioning --- p.161 / Chapter 5.8.4.4.3 --- Staining of sections --- p.162 / Chapter 5.8.4.4.4 --- Image analysis --- p.164 / Chapter 5.8.5 --- Statistical analysis --- p.165 / Chapter 5.9 --- RESULTS --- p.166 / Chapter 5.9.1 --- Effect of ELP and CDNR on bone micro-architecture --- p.and / Chapter bone --- density at the bone defect site --- p.166 / Chapter 5.9.2 --- Histomorphometrical findings in treatment of bone healing --- p.172 / Chapter 5.9.3 --- Effect of ELP and CDNR on biomechanics of bone --- p.175 / Chapter 5.9.4 --- Effect of ELP and CDNR on bone turnover --- p.178 / Chapter 5.10 --- DISCUSSION --- p.184 / Chapter CHAPTER 6: --- GENERAL DISCUSSION AND CONCLUSION --- p.193 / Chapter 6.1 --- UNKNOWN AREAS FOR THE STUDY OF ELP --- p.193 / Chapter 6.2 --- SUMMARY OF CRUCIAL FINDINGS OF THE OSTEOGENIC EFFECTS OF ELP IN EACH PART OF THIS STUDY --- p.194 / Chapter 6.2.1 --- Part 1: in vitro seropharmacological study on osteoporosis --- p.194 / Chapter 6.2.2 --- Part 2: in vivo study on disuse osteoporosis --- p.195 / Chapter 6.2.3 --- Part 3: in vivo study on bone healing --- p.196 / Chapter 6.3 --- COMMON OSTEOGENIC EFFECT OF ELP IN THE THREE PARTS OF THE WHOLE STUDY --- p.197 / Chapter 6.4 --- LIMITATIONS OF THE PRESENT STUDY --- p.197 / Chapter 6.5 --- SIGNIFICANCES OF THIS STUDY --- p.199 / Chapter 6.6 --- FUTURE STUDIES --- p.199 / BIBLIOGRAPHY --- p.201

Le but de cette thèse était d'explorer les relations entre le niveau de performance physique, le niveau d'activité physique usuel, les apports nutritionnels, les caractéristiques anthropométriques et le sommeil d'un côté et la densité minérale osseuse, la géométrie osseuse de la hanche et le trabecular bone score (TBS) de l'autre chez les jeunes adultes Libanais et Français agés de 18 à 35 ans. Dans un premier temps, nous avons mené 10 études préliminaires chez les jeunes adultes Libanais. Ces études ont montré que le poids, la masse maigre, la pratique d'activités physiques à impacts, la force maximale et le niveau de puissance musculaire sont des déterminants positifs de la DMO et que la pratique d'activités physiques à impats influence positivement les valeurs de TBS, les indices géométriques de résistance osseuse de la hanche (surface de la section transversale (CSA) et module de section (Z)) et les indices de résistance osseuse du col fémoral (Bending strength index (BSI), compressive strength index (CSI) et impact strength index (ISI)). Ces études préliminaires ont aussi montré des corrélations entre les apports protéiques et calciques d'une part et la DMO d'autre part chez les jeunes hommes. A la fin, une des études préliminaires a suggéré que la qualité de sommeil est associée à une meilleure DMO chez les jeunes hommes. Dans un deuxième temps, nous avons mené une étude sur 535 jeunes adultes français (342 femmes et 193 hommes) afin d'explorer les relations entre le niveau de performance physique, le niveau d'activité physique, la composition corporelle, les apports nutritionnels et la qualité du sommeil d'une part et les valeurs de BUA (Broadband Ultrasound Attenuation) d'autre part. Cette étude a montré que les caractéristiques anthropométriques (poids, IMC, masse grasse, tour de taille et tour de hanche) sont des facteurs corrélables à la BUA chez les femmes mais pas chez les hommes. D'autre part, il y avait une tendance de corrélation positive entre la performance en détente verticale et la BUA chez les hommes. En conclusion, cette thèse a pu définir un nombre important de facteurs corrélables à la BUA, à la DMO, à la géométrie osseuse et au TBS et a ainsi permis d'identifier de nouvelles relations entre les déterminants de santé et les paramètres osseux chez les jeunes adultes. / The aim of this study was to investigate the relations between physical performance level, physical activity level, nutritional intakes, anthropometrical characteristics and sleep on the one hand and bone mineral density, hip geometric indices and trabecular bone score (TBS) on the other hand in young Lebanese and French subjects aged to 18 to 35 years. In a first step, we led 10 preliminary studies in young Lebanese adults. These studies have shown that body weight, lean mass, high-impact physical activity practice, maximal strength and muscular power are positive determinants of bone mineral density, and that high-impact physical activity practice positively influences TBS values, geometric indices of hip bone strength (cross-sectional area (CSA) and section modulus (Z)) and femoral neck strength indices (Bending strength index (BSI), compressive strength index (CSI) and impact strength index (ISI)). These preliminary studies have also shown positive correlations between nutritional intakes (daily calcium intakes and daily protein intakes) and BMD values in young Lebanese men. Finally, one of these preliminary studies has shown that sleep quality is associated with a greater BMD in young men. In a second step, we led a study on 535 young French adults (342 women and 193 men) to explore the relations between physical performance level, physical activity level, nutritional intakes, anthropometrical characteristics and sleep on the one hand and BUA (Broadband ultrasound attenuation) values on the other hand. This study has showed that anthropometric characteristics (body weight, body mass index, waist circumference and hip circumference) are positively correlated to BUA values in women but not in men. On the other hand, we have noted a tendency of positive relation between vertical jump performance and BUA values in men. In conclusion, this thesis has defined an important number of factors significantly correlated to BUA, BMD, hip geometric indices and TBS and therefore permitted to identify new relations between health determinants and bone parameters in young adults.

Indiana University-Purdue University Indianapolis (IUPUI) / Mechanical loading associated with exercise is known to benefit bone health; however, most studies explore exercise benefits on bone mass independent of bone structure and strength. The purpose of this dissertation is to explore the response of the skeleton to exercise across the translational divide between animal- and human-based studies, with a particular emphasis on exercise-induced changes in bone structure and estimated strength. To explore the skeletal benefits of exercise, models were used wherein loading is introduced unilaterally to one extremity. Unilateral exercise enables the contralateral, non-exercised extremity to be used as an internal control site for the influences of systemic factors, such as genetics and circulating hormones. In study 1, a dose response between load magnitude and tibial midshaft cortical bone adaptation was observed in mice that had their right tibia loaded in axial compression at one of three load magnitudes for 3 d/wk over 4 weeks. In study 2, the ability of peripheral quantitative computed tomography to provide very good prediction of midshaft humerus mechanical properties with good short-term precision in human subjects was demonstrated. In study 3, collegiate-level jumping (long and/or high jump) athletes were shown to have larger side-to-side differences in tibial midshaft structure and estimated strength between their jump and lead legs than observed in non-jumping athletes. In study 4, prepubertal baseball players followed for 12 months were shown to gain more bone mass, structure and estimated strength in their throwing arm relative to their nonthrowing arm over the course of 12 months. These cumulative data using a combination of experimental models ranging from animal to cross-sectional and longitudinal human models demonstrate the ability of the skeleton to adapt its structure and estimated strength to the mechanical loading associated with exercise. Study of these models in future work may aid in optimizing skeletal responses to exercise.

University of Macau / Institute of Chinese Medical Sciences

Após a redução dos estrógenos no período da menopausa algumas mulheres passam a perder massa óssea acima de 1% ao ano chegando ao final de cinco anos com perda superior a 25%. Neste sentido, fatores como idade avançada, baixa ingestão de cálcio e menopausa precoce favorecem o aparecimento da osteoporose. Métodos preventivos como orientação nutricional para uma dieta adequada e o apoio da tecnologia por meio de aplicativos que avaliam o consumo alimentar são essenciais. Desta forma, objetivou-se neste estudo desenvolver um aplicativo em plataforma Android® voltado à avaliação das condições nutricionais e orgânicas envolvidas na saúde óssea e grau de risco para o desenvolvimento de osteoporose em mulheres pós-menopáusicas. Para o alcance deste objetivo procedeu-se um estudo com 72 mulheres com idade entre 46 a 79 anos, provenientes do programa de exercícios físicos para a saúde óssea do Laboratório de Pesquisas em Bioquímica e Densitometria da Universidade Tecnológica Federal do Paraná. Os dados foram coletados no segundo semestre de 2014 por meio de exames de Densitometria Óssea e Composição Corporal, Exames de Sangue, dados Antropométricos e Avaliação Nutricional. Foram incluídas no estudo mulheres com diagnóstico atualizado de osteopenia ou osteoporose primária, com idade igual ou superior a 45 anos em fase pós-menopáusica. Para a avaliação da densidade mineral óssea e composição corporal utilizou-se o aparelho de Absortometria de Dupla Energia de Raios-X (DXA) da marca HologicTM modelo Discovery A. Para a avaliação antropométrica foi incluído a Massa Corporal, Estatura, Circunferência Abdominal, Circunferência da Cintura e Circunferência do Quadril. O instrumento para avaliação de consumo alimentar utilizado foi o Recordatório de 24 horas de um dia (R24h). A estimativa da ingestão de energia e nutrientes foi realizada a partir da tabulação dos alimentos ingeridos no Software Diet Pro 4®. Em uma sub amostra com 30 mulheres com osteopenia/osteoporose foram realizados os exames de cálcio sérico e fosfatase alcalina. Os resultados demonstraram no grupo de mulheres (n=30) ingestão média de cálcio de 570mg/dia (±340). A análise do cálcio sérico apresentou média dentro da normalidade (10,20mg/dl±0,32) e valores médios e fosfatase alcalina ligeiramente aumentados (105,40 U/L±23,70). Ainda, houve importante correlação entre o consumo de ideal de proteínas e o consumo de cálcio diário (0,375 p valor 0,05). Com base nestes achados, foi desenvolvido um aplicativo fase inicial na plataforma Android® do sistema operacional do Google®, sendo denominado de OsteoNutri. Optou-se pela utilização Java Eclipse® onde nele foram executados a versão Android® do projeto; escolha de ícones de aplicação e configuração do editor visual para construção dos layouts do aplicativo. Foi utilizado o DroidDraw® para desenvolvimento das três interfaces gráficas do aplicativo. Para os testes práticos utilizou-se um celular compatível com a versão que foi criada (4.4 ou superior). O protótipo foi desenvolvido em conjunto com o Grupo de Desenvolvimento Aplicativos e Instrumentação (GDAI) da Universidade Tecnológica Federal do Paraná. Portanto, este aplicativo pode ser considerado uma importante ferramenta no controle dietético, possibilitando controle mais próximo de consumo de Cálcio e Proteínas dietéticas. / Following a drop in estrogen in the period of menopause some women begin to lose bone mass more than 1% per year reaching the end of five years with loss greater than 25%. In this regard, factors such as older age, low calcium intake and premature menopause favor the onset of osteoporosis. Preventive methods such as nutritional counseling to a proper diet and the support of technology through applications that assess dietary intake are essential. Thus, this study aimed to develop an application for Android® platform focused on the evaluation of nutritional and organic conditions involved in bone health and risks for developing osteoporosis in postmenopausal women. To achieve this goal we proceeded to a study of 72 women aged 46-79 years, from the physical exercise for bone health of the Laboratory for Research in Biochemistry and Densitometry the Federal Technological University of Paraná program. Data were collected in the second half of 2014 through tests Bone Densitometry and Body Composition, Blood Tests, Anthropometric data and Nutrition Assessment. The study included women with a current diagnosis of osteopenia or osteoporosis primary, aged more than 45 years postmenopausal. For the assessment of bone mineral density and body composition used the device Absorptiometry Dual Energy X-ray (DXA) brand Hologic Discovery TM Model A. For anthropometric assessment was included to body mass, height, abdominal circumference, Waist circumference and hip circumference. The instrument for assessing food consumption was used Recall 24 hours a day (24HR). The estimated intake of energy and nutrients was carried from the tabulation of the food eaten in the Software Diet Pro 4®. In a sub sample of 30 women with osteopenia / osteoporosis serum calcium and alkaline phosphatase tests were performed. The results demonstrated a group of women (n = 30) average calcium intake of 570mg / day (± 340). The analysis showed a mean serum calcium within the normal range (10,20mg / dl ± 0.32) and average values and slightly increased alkaline phosphatase (105.40 U / L ± 23.70). Furthermore, there was a significant correlation between the consumption of protein and the optimal daily intake of calcium (0.375 p-value 0.05). Based on these findings, we developed an application early stage in Android® platform operating system Google®, being called OsteoNutri. We chose to use Java Eclipse® where it was executed Android® version of the project; choice of application icons and setting the visual editor for building the application layouts. The DroidDraw® was used for development of the three application GUIs. For practical tests we used a cell compatible with the version that was created (4.4 or higher). The prototype was developed in conjunction with the Group and Instrumentation Applications Development (GDAI) of the Federal Technological University of Paraná. So this application can be considered an important tool in dietary control, allowing closer control consumption of calcium and dietary proteins.

Indiana University-Purdue University Indianapolis (IUPUI) / Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.

Indiana University-Purdue University Indianapolis (IUPUI) / An excess of bone resorption over bone formation leads to osteoporosis, resulting in a reduction of bone mass and an increase in the risk of bone fracture. Anabolic and anti-resorptive drugs are currently available for treatment, however, none of these drugs are able to both promote osteoblastogenesis and reduce osteoclastogenesis. This thesis focused on the role of eukaryotic translation initiation factor 2 alpha (eIF2alpha), which regulates efficiency of translational initiation. The elevation of phosphorylated eIF2alpha was reported to stimulate osteoblastogenesis, but its effects on osteoclastogenesis have not been well understood. Using synthetic chemical agents such as salubrinal and guanabenz that are known to inhibit the de-phosphorylation of eIF2alpha, the role of phosphorylation of eIF2alpha in osteoclastogenesis was investigated in this thesis. The questions addressed herein were: Does the elevation of phosphorylated eIF2alpha (p-eIF2alpha) by salubrinal and guanabenz alter osteoclastogenesis? If so, what regulatory mechanism mediates the process? It was hypothesized that p-eIF2alpha could attenuate the development of osteoclast by regulating the transcription factor(s) amd microRNA(s) involved in osteoclastogenesis. To test this hypothesis, we conducted in vitro and in silico analysis of the responses of RAW 264.7 pre-osteoclast cells to salubrinal and guanabenz. First, the in vitro results revealed that the elevated level of phosphorylated eIF2alpha inhibited the proliferation, differentiation, and maturation of RAW264.7 cells and downregulated the expression of NFATc1, a master transcription factor of osteoclastogenesis. Silencing eIF2alpha by RNA interference suppressed the downregulation of NFATc1, suggesting the involvement of eIF2alpha in regulation of NFATc1. Second, the in silico results using genome-wide expression data and custom-made Matlab programs predicted a set of stimulatory and inhibitory regulator genes as well as microRNAs, which were potentially involved in the regulation of NFATc1. RNA interference experiments indicated that the genes such as Zfyve21 and Ddit4 were primary candidates as an inhibitor of NFATc1. In summary, the results showed that the elevation of p-eIF2alpha by salubrinal and guanabenz leads to attenuation of osteoclastogenesis through the downregulation of NFATc1. The regulatory mechanism is mediated by eIF2alpha signaling, but other signaling pathways are likely to be involved. Together with the previous data showing the stimulatory role of p-eIF2alpha in osteoblastogenesis, the results herein suggest that eIF2alpha-mediated signaling could provide a novel therapeutic target for treatment of osteoporosis by promoting bone formation and reducing bone resorption.