FGFR 3 is one of a family of four high affinity receptors for FGF ligands. Activating mutations in FGFR 3 result in skeletal dysplasias that vary in severity from undetectable to neonatal lethal. Mice with congenital deficiency of FGFR3 develop severe kyphosis and skeletal overgrowth. FGFR3 is also expressed in calvarial pre-osteoblasts, osteoblast and articular chondrocytes, although it biological role in these cells remains undefined. By changing the genetic background of the Fgfr3-/- mice we were able to extend their lifespan and examine its impact on post-natal skeletal growth. To investigate the implication of FGFR 3 in post-natal cartilage and bone metabolism we used a combination of imaging, classic histology, molecular biology and biomechanical testing. The results demonstrated that the synovial joints of young adult Fgfr3-/- mice revealed a progressive deterioration, loss of the joint space width and changes in the subchondral bone. These alterations were accompanied by an increase of cartilage matrix degradation. Increased aggrecan and collagen type II degradation products, generated by MMPs were detected with DIAPEN and COL2-3/4C antibodies. Increased collagen type X, cellular hypertrophy and loss of proteoglycan at the articular surface were also demonstrated. A novel micro-mechanical indentation protocol revealed that the humeral heads of Fgfr3-/- mice were less stiff than those of wild type littermates. On the other hand, young adult Fgfr3-/- mice are osteopenic due to reduced cortical bone thickness and defective trabecular bone mineralization. The reduction in mineralized bone and lack of trabecular connectivity observed by micro-computed tomography were confirmed by histological and histomorphometric analyses, which revealed a significant decrease in calcein labeling of mineralizing surfaces and a significant increase in osteoid in the long bones of 4-month-old Fgfr3-/- mice. Primary cultures of adherent bone marrow-derived cells from Fgfr3-/- mice expressed markers of differentiated osteoblasts but developed fewer mineralized nodules than Fgfr3+/+ cultures of the same age. These data point to a major role for FGFR3 signaling in development and homeostatic maintenance of cartilage and bone post-natally and identify FGFR3 as a potential target for intervention in degenerative disorders of cartilage, osteopenia and those associated with defective bone mineralization.

No description available.

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

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Este trabalho tem por objetivo avaliar mulheres portadoras de endometriose em uso de anÃlogos de GnRH investigando o metabolismo Ãsseo e massa Ãssea atravÃs da dosagem de marcadores sÃricos e realizaÃÃo de ultra-sonometria do calcÃneo, respectivamente. Trata-se de estudo observacional transversal tipo caso â controle prospectivo. Foi desenvolvido na Maternidade-Escola Assis Chateaubriand (MEAC) â UFC. Foram avaliadas 99 mulheres, divididas em 3 grupos, sendo 32 portadoras de endometriose diagnosticada cirurgicamente e tratadas com goserelina 3,6mg SC a cada 28 dias (4 doses) â grupo endometriÃtico. O segundo grupo foi composto de 25 mulheres nÃo endometriÃticas e no menacme (controle negativo). O terceiro grupo foi composto de 42 mulheres nÃo endometriÃticas e menopausadas por no mÃnimo dois anos, os dois Ãltimos grupos sem uso de medicaÃÃes. Foi realizada avaliaÃÃo Ãssea atravÃs da ultra-sonometria do calcÃneo com o aparelho Achilles, da LunarÂ, sendo determinado o âstiffnessâ de cada grupo (uma combinaÃÃo de velocidade do som e grau de atenuaÃÃo ultra-sonogrÃfica), juntamente com as dosagens sÃricas de magnÃsio, fosfato, urÃia, creatinina, cÃlcio, fosfatase alcalina (FA), PTH, cortisol e hidroxiprolina, alÃm das dosagens de cÃlcio urinÃrio, cÃlcio urinÃrio/creatinina, e hidroxiprolina/creatinina. O grupo endometriÃtico somente foi submetido a esta avaliaÃÃo apÃs o uso da medicaÃÃo. A anÃlise estatÃstica foi realizada pelo programa SPSS for Windows 11.0.0. As dosagens de FA, cÃlcio urinÃrio e cÃlcio urinÃrio/creatinina foram semelhantes no grupo endometriÃtico (40,8Â7,7U/mL; 47,15Â10,8mmol/L; e 78,76Â23,0, respectivamente) e no grupo menopausado (38,65Â5,1U/mL; 36,8Â4,3mmol/L; e 55,21Â8,21, respectivamente) alÃm de significativamente superiores aos do grupo no menacme (28,5Â2,54U/mL; 26,4Â3,4mmol/L; e 39,52Â7,7, respectivamente). As dosagens de PTH do grupo endometriÃtico (23,99Â3,35nmol/L) foram semelhantes as das mulheres no menacme (29,15Â4,09nmol/L), ambas sendo significativamente menores que as mulheres menopausadas (41,14Â3,7nmol/L). As demais anÃlises foram semelhantes entre os grupos. Na avaliaÃÃo Ãssea o âstiffnessâ foi similar entre o grupo endometriÃtico (88,16Â2,86) e as mulheres menopausadas (83,70Â1,8), sendo ambos significativamente inferiores Ãs mulheres no menacme (97,02Â1,46). Conclui-se que as portadoras de endometriose apÃs tratamento com goserelina apresentaram intenso metabolismo Ãsseo e piora no padrÃo do tecido Ãsseo avaliada pela ultra-sonometria do calcÃneo aproximando-se do quadro encontrado em mulheres menopausadas hà pelo menos dois anos. NÃo se pode afirmar, no entanto, se tais alteraÃÃes sÃo devidas exclusivamente ao uso do anÃlogo do GnRH ou somam-se à prÃpria manifestaÃÃo da endometriose. Sugere-se que a endometriose e o uso de anÃlogos do GnRH sejam considerados como fatores de risco para o desenvolvimento de osteoporose, principalmente se associados a uma histÃria de uso crÃnico de corticÃides a qualquer Ãpoca da vida. / This research had as an objective of evaluate endometriotic women treated with GnRH analogues by investigating their bone turnover and bone structure using serum bone turnover markers and calcaneous ultrasonometry, respectively. This is a transversal, observational, prospective caseâcontrol study. It was developed at Maternidade-Escola Assis Chateaubriand (MEAC) â UFC. Ninety nine women, divided into three groups were analyzed. Thirty two endometriotic women were treated with goserelin 3,6mg SC 28/28d (4 doses) â Endometriotic group. Their disease had been confirmed by surgery. The second group had twenty five non endometriotic women and having menses (control group). The third group had 42 not endometriotic menopausal women, they were at menopause at least for 2 years. The latest two groups were not taking any treatment. The Achilles device from Lunar, had being used to analyse the bone structure through calcaneous ultrasonometry. We calculated the âstiffnessâ value for each group (a combination of sound velocity and ultrasonographic attenuation), and we also analysed the values of magnesium, phosphate, urea, creatinine, serum calcium, alkaline phosphatase (ALP), parathyroid hormone (PTH), cortisol, hydroxyproline, urinary calcium, urinary calcium/creatinine, and hydroxyproline/creatinine. The endometriotic group was evaluated only after the treatment. The statistical analysis had being done by SPSS program for Windows version 11.0.0. The values of ALP, urinary calcium and urinary calcium/creatinine were similar to endometriotic group (40.8Â7.7U/mL; 47.15Â10.8mmol/L; and 78.76Â23.0, respectively) and to menopausal group (38.65Â5.1U/mL; 36.8Â4.3mmol/L; and 55.21Â8.21, respectively) although significantly higher than control group (28.5Â2.54U/mL; 26.4Â3.4mmol/L; and 39.52Â7.7, respectively). The values of PTH from endometriotic group (23.99Â3.35nmol/L) were similar to control group (29.15Â4.09nmol/L), and both were significantly lower than menopausal one (41.14Â3.7nmol/L). The other values were equal between groups. At the evaluation of bone âstiffnessâ the values were similar between endometriotic (88.16Â2.86) and menopausal groups (83.70Â1.8), and both were significantly lower than control group (97.02Â1.46). Concluding the endometriotic women who received treatment with goserelin showed an intense bone metabolism and a bone deficit at calcaneous ultrasonometry almost like women at post-menopausal at least two years. Therefore, we can not affirm if these alterations were caused exclusively by the GnRH-analogue therapy or were influenciated by endometriosis itself. We suggest that endometriosis and treatment with GnRH analogues might be considered as risks factors for the development of osteoporosis, principally if they are associated with chronic corticoid treatment at any point in a lifetime.

O hormônio da paratireóide (PTH) tem sido utilizado como um agente anabólico ósseo para o tratamento de condições de osteopenia / osteoporose, prevenção e consolidação de fraturas. O papel do fator de crescimento semelhante à insulina I (IGF-I), como um potencial mediador dos efeitos anabólicos do PTH, é controverso. O rato dwarf pode ser adequado para o estudo dessas interações in vivo, uma vez que a os níveis séricos de hormônio do crescimento (GH) encontram-se reduzidos a cerca de 6% dos valores normais em fêmeas e os níveis séricos de IGF-I, a cerca de 10% dos valores normais, mas estes animais são saudáveis e sem malformações esqueléticas. Os objetivos deste estudo foram: 1 - Avaliar o rato dwarf (dw-/dw-) como um modelo animal para o estudo dos efeitos da deficiência do GH e do IGF-I sobre o esqueleto e o metabolismo ósseo; 2 - Comparar os efeitos do tratamento com PTH sobre o esqueleto e formação óssea em ratos dwarf e em ratos Lewis, sua linhagem de origem. A partir de 9 semanas de idade, ratas Lewis e dwarf receberam injeções por via subcutânea, diariamente, por duas semanas, com medicamento placebo ou fragmento de hormônio paratireóideo humano, hPTH 1-34, na dose de 50 g / kg de peso corpóreo (N = 7-13/grupo). Foram realizadas avaliações do peso corpóreo semanalmente e, por ocasião da eutanásia, na 11ª semana, foram coletadas amostras de sangue para realização de dosagens séricas de IGF-I (ELISA). As vértebras lombares e as metáfises proximais das tíbias foram avaliadas por meio de histomorfometria óssea. Os fêmures direitos foram mensurados e analisados por tomografia quantitativa periférica computadorizada (pQCT). Os níveis séricos de IGF-I mostraram-se três vezes menores nas ratas dwarf quando comparados aos observados nas ratas Lewis, a despeito do tratamento com PTH, que não provocou aumento de IGF-I em nenhum dos dois grupos. No entanto, o PTH aumentou significativamente o volume ósseo trabecular em ambos os grupos, dwarf (p<0.003) e Lewis (p < 0.0001) comparados aos seus respectivos grupos controle, efeito associado ao aumento da espessura e da distância trabeculares. As ratas dwarf tratadas com PTH também exibiram aumentos de 7 a 13 vezes na superfície de mineralização e na taxa de formação óssea respectivamente, quando comparadas às ratas dwarf tratadas com placebo, enquanto as ratas Lewis tratadas com PTH mostraram aumentos de 3 e 4 vezes quando comparadas as ratas Lewis tratadas com placebo. A taxa de aposição mineral, indicativa de atividade osteoblástica, estava aumentada nas ratas dwarf e Lewis tratadas com PTH (p<0.0001) comparadas aos seus respectivos grupos controle. As análises pela pQCT das metáfises femorais distais revelaram que todos os parâmetros estruturais do osso trabecular (BMC total, BMD total, BMC trabecular e BMD trabecular) também apresentaram valores significativamente aumentados nas ratas, Lewis e dwarf, tratadas com PTH, quando comparadas às ratas tratadas com placebo (p<0.0001). Ao se considerar os parâmetros para o osso cortical, praticamente todos os valores obtidos nas diáfises femorais (BMC total, BMD total, BMC cortical, BMD cortical, área cortical, espessura cortical, circunferência periosteal e endosteal) não mostraram qualquer efeito do tratamento com PTH nos dois grupos. Em conclusão, o PTH induziu efeitos anabólicos altamente significativos no tecido ósseo trabecular das tíbias e vértebras lombares, a despeito dos baixos níveis circulantes de IGF-I em animais da linhagem dwarf. A resposta positiva ao tratamento com PTH confirma a sua utilização terapêutica como potente agente anabólico ósseo mesmo em face à deficiência no eixo GH/IGF-I / The parathyroid hormone (PTH) has been used as a bone anabolic agent to treat osteopenic/osteoporotic conditions, prevention and healing of fractures. The role of insulin-like growth factor I (IGF-I) as a potential mediator for the bone anabolic effects of PTH is controversial. The dwarf rat (dw-/dw-) may be suitable to study these interactions in vivo, since GH synthesis is selectively reduced to about 6% of normal in females, and serum IGF-I levels are about 10% of normal, but these animals are healthy without skeletal malformations. The objectives of this study were: 1- Evaluate the dwarf rat (dw-/dw-) as an animal model for studies of the effects of GH and IGF-I deficiency on the skeleton and bone metabolism; 2- Compare the skeletal effects of PTH treatment in dwarf rats and their background strain, Lewis rats. At 9 weeks of age, female Lewis and dwarf rats were injected SC daily for 2 weeks with vehicle or human parathyroid hormone fragment, hPTH 1-34, at a dose of 50 g/kg body weight (N=7- 13/group). The body weight was evaluated weekly and at the time of euthanasia, at 11 weeks, blood samples were collected. Serum IGF-I was measured by ELISA, and cancellous bone histomorphometry was performed in the lumbar vertebral body and tibial proximal metaphysis. The right femurs were measured, scanned and analyzed by peripheral quantitative computed tomography (pQCT). Serum levels of IGF-I were nearly 3-fold lower in dwarf rats compared with Lewis rats regardless of treatment, but PTH treatment did not increase serum IGF-I in either Lewis or dwarf rats. However, PTH significantly increased cancellous bone volume in both dwarf (P<0.003) and Lewis rats (P<0.0001) when compared to vehicle-treated rats, which was associated with increased trabecular width and decreased trabecular separation. PTH-treated dwarf rats also exhibited 7- and 13-fold increases in mineralizing surface and bone formation rate respectively, compared to vehicle-treated dwarf rats, while PTH-treated Lewis rats showed 3- and 4-fold increases when compared to vehicle-treated Lewis rats. Mineral apposition rate, an index of osteoblast activity, was increased in PTH-treated dwarf rats (P<0.0001) and in Lewis rats (P<0.0001) compared to their respective control groups. The pQCT analyses of the distal femoral metaphysis revealed that cancellous bone structural parameters (total BMC, total BMD, trabecular BMC, and trabecular BMD) also presented significantly higher values in PTH-treated dwarf and Lewis rats, when compared to vehicle treated rats (P<0.0001). When considering cortical bone parameters, almost all the values obtained at the femoral shafts (total BMC, total BMD, cortical BMC, cortical area, cortical thickness, periosteal and endocortical circumferences) did not show any PTH treatment effect in either groups. In conclusion, PTH induced highly significant anabolic effects in vertebral and tibial cancellous bone despite low circulating levels of IGF-I in dwarf rats. The positive response to PTH treatment confirms its therapeutic use as a potent bone anabolic agent, even in the face of GH/IGF-I deficiency

O hormônio da paratireóide (PTH) tem sido utilizado como um agente anabólico ósseo para o tratamento de condições de osteopenia / osteoporose, prevenção e consolidação de fraturas. O papel do fator de crescimento semelhante à insulina I (IGF-I), como um potencial mediador dos efeitos anabólicos do PTH, é controverso. O rato dwarf pode ser adequado para o estudo dessas interações in vivo, uma vez que a os níveis séricos de hormônio do crescimento (GH) encontram-se reduzidos a cerca de 6% dos valores normais em fêmeas e os níveis séricos de IGF-I, a cerca de 10% dos valores normais, mas estes animais são saudáveis e sem malformações esqueléticas. Os objetivos deste estudo foram: 1 - Avaliar o rato dwarf (dw-/dw-) como um modelo animal para o estudo dos efeitos da deficiência do GH e do IGF-I sobre o esqueleto e o metabolismo ósseo; 2 - Comparar os efeitos do tratamento com PTH sobre o esqueleto e formação óssea em ratos dwarf e em ratos Lewis, sua linhagem de origem. A partir de 9 semanas de idade, ratas Lewis e dwarf receberam injeções por via subcutânea, diariamente, por duas semanas, com medicamento placebo ou fragmento de hormônio paratireóideo humano, hPTH 1-34, na dose de 50 g / kg de peso corpóreo (N = 7-13/grupo). Foram realizadas avaliações do peso corpóreo semanalmente e, por ocasião da eutanásia, na 11ª semana, foram coletadas amostras de sangue para realização de dosagens séricas de IGF-I (ELISA). As vértebras lombares e as metáfises proximais das tíbias foram avaliadas por meio de histomorfometria óssea. Os fêmures direitos foram mensurados e analisados por tomografia quantitativa periférica computadorizada (pQCT). Os níveis séricos de IGF-I mostraram-se três vezes menores nas ratas dwarf quando comparados aos observados nas ratas Lewis, a despeito do tratamento com PTH, que não provocou aumento de IGF-I em nenhum dos dois grupos. No entanto, o PTH aumentou significativamente o volume ósseo trabecular em ambos os grupos, dwarf (p<0.003) e Lewis (p < 0.0001) comparados aos seus respectivos grupos controle, efeito associado ao aumento da espessura e da distância trabeculares. As ratas dwarf tratadas com PTH também exibiram aumentos de 7 a 13 vezes na superfície de mineralização e na taxa de formação óssea respectivamente, quando comparadas às ratas dwarf tratadas com placebo, enquanto as ratas Lewis tratadas com PTH mostraram aumentos de 3 e 4 vezes quando comparadas as ratas Lewis tratadas com placebo. A taxa de aposição mineral, indicativa de atividade osteoblástica, estava aumentada nas ratas dwarf e Lewis tratadas com PTH (p<0.0001) comparadas aos seus respectivos grupos controle. As análises pela pQCT das metáfises femorais distais revelaram que todos os parâmetros estruturais do osso trabecular (BMC total, BMD total, BMC trabecular e BMD trabecular) também apresentaram valores significativamente aumentados nas ratas, Lewis e dwarf, tratadas com PTH, quando comparadas às ratas tratadas com placebo (p<0.0001). Ao se considerar os parâmetros para o osso cortical, praticamente todos os valores obtidos nas diáfises femorais (BMC total, BMD total, BMC cortical, BMD cortical, área cortical, espessura cortical, circunferência periosteal e endosteal) não mostraram qualquer efeito do tratamento com PTH nos dois grupos. Em conclusão, o PTH induziu efeitos anabólicos altamente significativos no tecido ósseo trabecular das tíbias e vértebras lombares, a despeito dos baixos níveis circulantes de IGF-I em animais da linhagem dwarf. A resposta positiva ao tratamento com PTH confirma a sua utilização terapêutica como potente agente anabólico ósseo mesmo em face à deficiência no eixo GH/IGF-I / The parathyroid hormone (PTH) has been used as a bone anabolic agent to treat osteopenic/osteoporotic conditions, prevention and healing of fractures. The role of insulin-like growth factor I (IGF-I) as a potential mediator for the bone anabolic effects of PTH is controversial. The dwarf rat (dw-/dw-) may be suitable to study these interactions in vivo, since GH synthesis is selectively reduced to about 6% of normal in females, and serum IGF-I levels are about 10% of normal, but these animals are healthy without skeletal malformations. The objectives of this study were: 1- Evaluate the dwarf rat (dw-/dw-) as an animal model for studies of the effects of GH and IGF-I deficiency on the skeleton and bone metabolism; 2- Compare the skeletal effects of PTH treatment in dwarf rats and their background strain, Lewis rats. At 9 weeks of age, female Lewis and dwarf rats were injected SC daily for 2 weeks with vehicle or human parathyroid hormone fragment, hPTH 1-34, at a dose of 50 g/kg body weight (N=7- 13/group). The body weight was evaluated weekly and at the time of euthanasia, at 11 weeks, blood samples were collected. Serum IGF-I was measured by ELISA, and cancellous bone histomorphometry was performed in the lumbar vertebral body and tibial proximal metaphysis. The right femurs were measured, scanned and analyzed by peripheral quantitative computed tomography (pQCT). Serum levels of IGF-I were nearly 3-fold lower in dwarf rats compared with Lewis rats regardless of treatment, but PTH treatment did not increase serum IGF-I in either Lewis or dwarf rats. However, PTH significantly increased cancellous bone volume in both dwarf (P<0.003) and Lewis rats (P<0.0001) when compared to vehicle-treated rats, which was associated with increased trabecular width and decreased trabecular separation. PTH-treated dwarf rats also exhibited 7- and 13-fold increases in mineralizing surface and bone formation rate respectively, compared to vehicle-treated dwarf rats, while PTH-treated Lewis rats showed 3- and 4-fold increases when compared to vehicle-treated Lewis rats. Mineral apposition rate, an index of osteoblast activity, was increased in PTH-treated dwarf rats (P<0.0001) and in Lewis rats (P<0.0001) compared to their respective control groups. The pQCT analyses of the distal femoral metaphysis revealed that cancellous bone structural parameters (total BMC, total BMD, trabecular BMC, and trabecular BMD) also presented significantly higher values in PTH-treated dwarf and Lewis rats, when compared to vehicle treated rats (P<0.0001). When considering cortical bone parameters, almost all the values obtained at the femoral shafts (total BMC, total BMD, cortical BMC, cortical area, cortical thickness, periosteal and endocortical circumferences) did not show any PTH treatment effect in either groups. In conclusion, PTH induced highly significant anabolic effects in vertebral and tibial cancellous bone despite low circulating levels of IGF-I in dwarf rats. The positive response to PTH treatment confirms its therapeutic use as a potent bone anabolic agent, even in the face of GH/IGF-I deficiency

<p>Uvod: Hepatička osteodistrofija je termin koji obuhvata metaboličke bolesti kosti udružene sa hroničnim bolestima jetre. U alkoholnoj cirozi (AC) jetre postoji visoka zastupljenost deficijencije vitamina D proporcionalna stepenu disfunkcije jetre, ali njena uloga u patogenezi hepatičke osteodistrofije nije dovoljno obja&scaron;njena. Nivo 25(OH)D odražava status vitamina D. Kod AC jetre izmenjena je metabolička aktivnost kosti i suprimirano je formiranje kosti &scaron;to dovodi do smanjenja ko&scaron;tane mase. U centru interesovanja je postizanje optimalnog statusa vitamina D. Stavovi o suplementaciji vitaminom D kod AC jetre nisu jasno definisani. Cilj rada: Utvrditi nivo vitamina D, ispitati metaboličku aktivnost kosti i mineralnu gustinu kosti kod bolesnika sa AC jetre. Utvrditi efekte suplementacije sa 1000 IU vitamina D3 na dan tokom godinu dana u odnosu na metaboličku aktivnost kosti i mineralnu gustinu kosti kod ispitivanih bolesnika. Bolesnici i metode: Istraživanje je sprovedeno na Klinici za gastroenterologiju i hepatologiju Kliničkog centra Vojvodine u Novom Sadu kao prospektivna intervencijska studija sa primenom suplementacije sa 1000 IU vitamina D3 na dan kod bolesnika sa AC jetre. Grupu bolesnika koja je uključena u istraživanje (1) činilo je 70 bolesnika mu&scaron;kog pola sa dijagnozom AC jetre. Bolesnici su imali četiri pregleda (P), odnosno tačke studije: P1-uključivanje bolesnika i započinjanje suplementacije vitaminom D; P2, P3 i P4 posle tri, &scaron;est i dvanaest meseci suplementacije vitaminom D, redom. Prilikom svakog pregleda rađene su analize funkcije jetre, metabolizma kosti i statusa vitamina D. Na početku (P1) i na kraju istraživanja (P4) vr&scaron;eno je merenje mineralne gustine kosti (BMD) DXA metodom. Gubitak bolesnika od P1 do P4 bio je dvadeset, na različitim tačkama studije. Prvi deo istraživanja odnosi se na Grupu bolesnika koja je uključena u istraživanje (1) i zavr&scaron;ila prvi pregled (P1). Pedeset bolesnika je zavr&scaron;ilo kompletno istraživanje po predviđenom protokolu i oni se zbog realizacije svih pregleda i ponovljenih merenja posmatraju kao: Grupa bolesnika koja je zavr&scaron;ila istraživanje (2). Rezultati: (1): Kod bolesnika sa AC jetre utvrđena je deficijencija vitamina D, snižen nivo osteokalcina, normalni nivoi CrossLapsa, PTH, ukupnog i jonizovanog kalcijuma, fosfora i magnezijuma. Osteopeniju je imalo 42,65% a osteoporozu 14,71% ispitanika. Kod svih ispitanika najniži BMD izmeren je na vratu femura. (2): Suplementacija vitaminom D dovela je do značajnog porasta 25(OH)D. U odnosu na osteokalcin konstatovana je pozitivna razlika vrednosti P1/P4, iako je nivo ostao ispod donje granice normale. Kod nivoa CrossLapsa i PTH razlika P1/P4 je negativna, ali su nivoi u sva četiri merenja u okviru referentnih vrednosti. Na lumbalnoj kičmi do&scaron;lo je do pobolj&scaron;anja BMD za 0.87%, a pogor&scaron;anja su na vratu femura -1.87 % i kuku -1.65%. Konstatovano je i pobolj&scaron;anje funkcije jetre. Zaključci: Kod bolesnika sa AC jetre pobolj&scaron;anje statusa vitamina D dovodi do povećanja formiranja kosti i pobolj&scaron;anja ko&scaron;tane mase na lumbalnoj kičmi. Neophodno je određivanje statusa vitamina D kod svih bolesnika sa AC jetre i uvođenje suplementacije vitaminom D kod bolesnika koji imaju nivo 25(OH)D &lt; 80 nmol/l, uz tromesečne kontrole efekta. Kod postavljanja dijagnoze AC jetre potrebno je inicijalno određivanje BMD. Kod suplementacije vitaminom D nakon inicijalnog DXA pregleda sledeći se preporučuje nakon jedne do dve godine.</p> / <p>Introduction: The term Hepatic osteodystrophy defines a group of metabolic bone diseases associated with underlying chronic liver disease. Alcoholic liver cirrhosis (ALC) is characterized by high incidence of vitamin D deficiency that is proportional to the level of liver failure; however, its role in the pathogenesis of hepatic osteodystrophy has not yet been fully elucidated. The level of 25(OH)D best reflects the vitamin D status. ALC is characterized by changed bone metabolic activity and suppressed bone formation, resulting in the decrease in bone mass. The key topic of interest is the achievement of optimal vitamin D status. The attitude of health professionals towards vitamin D supplementation in alcoholic liver cirrhosis has not yet been clearly defined. The aim of the research: Determining of vitamin D levels, investigating the metabolic activity of the bone and bone mass in patients with alcoholic liver cirrhosis (ALC); Determining the effects of vitamin D3 supplementation at the dose 1000 IU/day during a one-year period in relation to metabolic activity of the bone and bone mineral density (BMD) in the investigated patient population. Patients and methods: The research was conducted at the Clinic for Gastroenterology and Hepatology of the Clinical Centre of Vojvodina in Novi Sad. The research was designed as a prospective interventional study implicating vitamin D3 supplementation at the dose 1000 IU/day to patients with ALC. The investigated patient population (1) encompassed 70 male patients diagnosed with ALC. The patients underwent four examinations (P), that is, research phases: P1 &ndash; inclusion of the patient into the study and introduction of vitamin D supplementation; P2, P3 and P4 after 3, 6 and 12 months of vitamin D supplementation treatment, respectively. Each examination included the analysis of liver function, bone metabolism and vitamin D status. At the beginning (P1) and at the end (P4) of the investigation period, bone mineral density (BMD) was measured by means of dual-energy x-ray absorptiometry (DXA) method. Twenty patients dropped out from the research at different stages throughout the investigation period (P1 to P4). The first part of the investigation pertains to the Group of patients who were included into the study (1) and completed the first examination (P1). Fifty patients have completed the entire research according to the foreseen protocol encompassing all examinations and repeated measurements. These patients are considered a Group of patients who completed the research (2) Results: (1): In ALC patients, vitamin D deficiency and decreased osteocalcin levels were established, as well as normal levels of CrossLaps, PTH, total and ionized calcium, phosphorus and magnesium. Osteopenia and osteoporosis were established in 42.65% and 14.71% of patients, respectively. The lowest BMD was measured in the femoral neck in all patients. (2): Vitamin D supplementation resulted in significant increase in 25(OH)D. Analysis of osteocalcin level revealed positive P1/P4 difference, even though the level remained below the lower normal limit. The levels of CrossLaps and PTH revealed negative P1/P4 difference; however, the levels determined at all four measurements were within the reference values. An improvement of BMD for 0.87% was established in lumbar spine, whereas a decrease was noticed in femoral neck (1.87%) and hip (1.65%). Furthermore, an improvement of liver function was established. Conclusions: Improvement of vitamin D status in ALC patients results in an increase of bone formation and improvement of body mass in lumbar spine. Determining the vitamin D status in all patients with ALC is of outmost importance, as well as the vitamin D supplementation of patients with levels of 25(OH)D &lt; 80 nmol/l along with the monitoring of treatment outcome at three-month intervals. Establishment of the diagnosis of alcoholic liver cirrhosis should encompass initial measurement of BMD. In case of vitamin D supplementation treatment, the initial DXA examination should be repeated after the period of one to two years.</p>