Abstract Whenever a foreign material is implanted into a human body an implant–tissue interface area forms between them. In this microenvironment, interactions take place between the implant and the surrounding tissue. The implantation of a biomaterial into tissue results in injury and initiation of the inflammatory response. This host response to biomaterials is an unavoidable series of events that occur when tissue homeostasis is disturbed by the implantation process. In bone tissue, biocompatible implants must initially be capable of strong bone implant contact and subsequently, allow the normal bone remodeling cycle around the implant. NiTi is a metal alloy composed of approximately a 50:50 ratio of nickel and titanium. It possesses shape memory and superelasticity properties, which make it an interesting biomaterial. NiTi has two phases: austenite and martensite. A decrease in temperature or applied stress induce the austenite-to-martensite transformation. Heating or removing the stress restores the parent austenite phase. The alloy in its martensite structure can be reshaped and strained several times more than a conventional metal alloy without irreversible deformation of the material. The alloy returns to its original shape as it changes from martensite-to-austenite. This transformation is seen as the macroscopic shape memory effect. This study further investigated the biocompatibility of NiTi, especially the bone cell response to both austenite and martensite. Different surface treatments were investigated in order to improve and possibly even control NiTi's bioactivity as a bone implant material. Osteoclasts grew and attached well on the austenite NiTi phase, but the results indicated that the biocompatibility of martensite NiTi was compromised. Oxidation of the NiTi surface improved osteoblast attachment and viability. This was due to the formation of a TiO2 surface layer of moderate thickness. Coating the NiTi surface with the extracellular matrix protein fibronectin was shown to enhance osteoblast proliferation and increase the number of cells in the G1 cell cycle stage. Austenite was more prone to show these effects than martensite. A sol-gel derived titania-silica surface treatment was observed to increase the bone implant contact of functional NiTi intramedullary nails. The surface treatment was most effective with the constant bending load provided by the NiTi nail.

Abstract Mammalian extracellular matrix metalloproteinases, MMPs, are a family of enzymes capable of degrading components of the connective tissue. The in vivo regulation of the cell lineage-specific expression of MMPs, however, is not well known. This study used transgenic mice to identify cell-specific elements in the upstream regulatory regions of MMP-2 and MMP-9. Transgenic mice were generated by pronuclear microinjections into fertilised oocytes using lacZ as a reporter gene. The reporter gene constructs containing varying lengths of the MMP-9 5'-upstream region revealed an area that allowed for expression in osteoclasts and migrating keratinocytes, the cells that also express MMP-9 in vivo. The sequence driving the cell specific expression included the nucleotides from -2722 to -7745. When the same upstream regulatory fragment of MMP-9 was used to drive the expression of the human tissue specific inhibitor of MMPs, TIMP-1, instead of lacZ, the transgenic mice developed normally and the animals were fertile with normal post-embryonic growth. However, cutaneous wound healing was remarkably retarded, but not totally prevented, and the migration of keratinocytes over the wound was slow. The mice expressed the human TIMP-1 in keratinocytes during wound healing and in situ zymography revealed a total blockage of the gelatinolytic activity of MMP-2 and MMP-9, the main gelatinases active in the healing wound tissues. By using a sequence of 6500 base pairs from the 5'-upstream regulatory region of the MMP-2 gene it was possible to drive the expression of lacZ in mesenchymal cells of the developing transgenic mouse embryo. The expression pattern was similar to that found in previous in situ hybridization studies, following the different stages of tissue morphogenesis and being present in the areas of basement membrane degradation and epithelial cell invasion. Computer analyses of the sequence revealed three regulatory upstream regions conserved between human, mouse, and rat, and possibly responsible for the cell-and tissue specificity. New transgene constructs containing fragments of the conserved regions will provide a more detailed profile of the in vivo MMP-2 regulation in the future. This study defined a fragment in the upstream regulatory region of MMP-9 that is essential for expression in osteoclasts and migrating keratinocytes. Furthermore, the keratinocyte derived MMPs, including MMP-9, were found to play important role in epithelial cell migration in the area of the healing wound.

L’ostéoporose touche principalement les femmes après la ménopause, c’est une maladie caractérisée par une détérioration de la minéralisation et de la micro-architecture de l’os. L’objectif du travail de thèse présenté ici est d’identifier une fraction protéique laitière ayant un effet stimulant sur la formation osseuse. Une telle fraction, ajoutée dans un produit ou un complément alimentaire, pourrait contribuer à réduire la perte osseuse. La première étape du projet consiste à produire les fractions laitières. Des protéines laitières (caséine ou protéines sériques) ont été digérées par des enzymes puis filtrées pour les fractionner selon leur poids moléculaire. Les fractions obtenues ont ensuite été testées sur des cultures primaires de cellules osseuses. Certaines fractions protéiques laitières ont augmenté la prolifération et la différenciation des ostéoblastes. Parmi ces fractions actives, la fraction correspondant au rétentat d’une filtration sur un filtre à 10kDa d’un hydrolysat de caséine par de la chymotrypsine a été sélectionnée pour être testée sur animaux. Cette fraction a été nommée fraction CR10. Pour étudier l’activité du CR10 in vivo sur le métabolisme osseux, un modèle de souris sous restriction protéique est mis au point. Nos études démontrent que, lorsque le régime est basé sur des protéines de soja, le passage d’un régime contenant 20% de protéines à un régime contenant 6% de protéines induit une réduction de la formation osseuse. Le traitement des souris sous restriction protéique avec du CR10 n’a eu aucun effet, ce qui signifie que le CR10 n’arrive pas à exercer son activité anabolique in vivo. En revanche, si de la caséine est donnée à la place du soja ou si de la PTH est injectée aux souris, la formation osseuse est augmentée. Ces résultats suggèrent que la fraction CR10 n’est pas un bon candidat comme fraction anabolique. En revanche, l’effet positif de la caséine par rapport au soja pourrait être exploité lors de futures études visant à mettre au point une fraction caséique ostéoanabolique. / Osteoporosis is a disease mainly affecting women after menopause, characterized by a reduced bone mineralization and a deterioration of bone micro-architecture. The aim of this thesis is to identify a milk protein fraction able to stimulate bone formation. When added to a food product, this fraction could reduce bone loss. The first task of this project was to produce the milk protein fractions. Milk proteins (casein or whey proteins) were digested by enzymes and fractionated by filtration according to their molecular weight. The fractions obtained were then tested on primary cultures of bone cells. Some of the milk protein fractions tested were able to increase proliferation and differentiation of osteoblasts. Among these active fractions, the one obtained by digestion of casein by chymotrypsin followed by filtration through a 10 kDa filter have been selected to be tested on animals. This fraction is named CR10. To study the activity of CR10 in vivo, a protein-restricted mouse model has been developed. Our studies showed that a reduction of protein in the diet from 20% to 6% impaired bone formation when the diet was based on soy protein. When these protein-restricted mice ingested the CR10 fraction, no improvement of the BMD was reported, which means that the CR10 cannot exert its anabolic activity in vivo. However, if casein is given instead of soy or if PTH is injected to the mice, bone formation is increased. These results suggest that the CR10 is not a good candidate as an anabolic fraction. However, the positive effect of casein compared to soy could be exploited in future studies aimed at finding an osteoanabolic casein fraction.

Orientador: Pedro Paulo Chaves de Souza / Resumo: A periodontite é resultado do desequilíbrio entre o biofilme bacteriano e a resposta imune do hospedeiro. Componentes bacterianos, como o lipopolissacarídeo (LPS) e as lipoproteínas, são reconhecidos pelo sistema imune e desencadeiam a produção de citocinas que auxiliam no combate à infecção, mas também induzem a destruição tecidual. A participação do LPS na destruição óssea já é bem estabelecida, porém o papel das lipoproteínas na periodontite permanece carente de investigação. Na periodontite, citocinas pró-inflamatórias participam do processo de destruição do tecido ósseo. Neste processo, são secretadas também citocinas osteoprotetoras. Dentre elas, a interleucina 4 (IL-4) é reconhecida pela propriedade de inibir a produção citocinas pró inflamatórias como IL-1, IL-6 e TNF-α. O papel protetor de IL-4 na osteoclastogênese e na doença periodontal induzida por lipoproteína ainda não foi investigado. Nosso estudo foi divido em dois capítulos. No capítulo 1, hipotetizamos que a lipoproteína sintética Pam2CSK4 (PAM2) poderia induzir a reabsorção óssea periodontal. Para isso, foram utilizados camundongos C57bl/6, que receberam injeções a cada 2 dias, por 24 dias, do veículo, LPS de Escherichia. coli ou PAM2, entre o primeiro e segundo molar superior. Após o período experimental, os animais foram eutanasiados e destinados à análise por microCT, análise histológica e imunohistoquímica para marcação dos osteoclastos. A PAM2 apresentou a capacidade de induzir a perda óssea alveolar, ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The pathogenesis of periodontitis is a result of imbalance between the bacterial biofilm and the host immune response. Bacterial components such as lipopolysaccharide (LPS) and lipoproteins, activate the immune system leading to periodontal distruction. The participation of LPS in periodontal bone destruction is well established, but there is a lack of information about the role of lipoproteins in periodontitis. In the pathogenesis of periodontitis, these molecular patterns are recognized by host immune system and trigger the production of cytokines that participate of antimicrobial response, but also induce tissue destruction. On the other hand, antinflamatory cytokines produced by Th2 cells, such as IL-4, have an osteoprotective phenotype. The role of IL-4 in lipoprotein-induced periodontitis was not yet investigated. Thus, this thesis was divided in two chapters. In chapter 1, we investigated the role of lipoproteins in the pathogenesis of periodontitis in mice. In this study, we hypothesized that the synthetic lipoprotein Pam2CSK4 (PAM2) can induce periodontal bone resorption. C57bl / 6 mice received bilateral injections every other day for 24 days of: vehicle, Escherichia coli LPS or PAM2, between the first and second upper molars. Twenty-four hours after the last injection, the mice were euthanized and the jaw bones were scanned for micro computed tomography, decalcified and processed for histological analysis and stained for tartrate-resistant acid phosphatase, phenoty... (Complete abstract click electronic access below) / Doutor

Orientador: Morgana Rodrigues Guimarães Stabili / Resumo: As chalconas são um grupo de compostos fenólicos derivados de plantas, com diversas propriedades biológicas. Em função dos seus efeitos farmacológicos, diferentes derivados chalcônicos sintéticos, baseados em seus análogos naturais, têm sido investigados como agentes terapêuticos. Estudos clínicos e pré-clínicos têm demonstrado sua efetividade no tratamento de doenças inflamatórias como câncer e artrite, e em patologias ósseas como osteoporose e tumores ósseos. Considerando suas propriedades biológicas e com objetivo de identificar compostos que possam atuar no tratamento de doenças ósseo inflamatórias, avaliamos a habilidade de um novo composto chalcônico, a Chalcona T4, de suprimir a inflamação e osteoclastogênese em um modelo de doença periodontal. No estudo in vivo, um ensaio de toxicidade em camundongos demonstrou que a administração de diferentes doses da chalcona T4 (5, 50, 100 e 200 mg/kg) por um período de 15 dias, via intragástrica, diariamente, não causou alterações físicas ou comportamentais. Análise histopatológica do rim, fígado e estômago destes animais também indicaram ausência de toxicidade em todas as doses avaliadas. Determinada a ausência de toxicidade, periodontite foi experimentalmente induzida em ratos pela colocação de ligaduras ao redor dos primeiros molares inferiores. Chalcona T4 foi administrada diariamente por gavagem intragástrica em duas doses (5 e 50 mg/kg) durante 15 dias. Ao final do período experimental, as amostras contendo tecido gengival ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Les maladies inflammatoires chroniques sont associées à un maintien de la réponse immunitaire et notamment la présence de cellules T CD4+ mémoires. Les maladies inflammatoires chroniques de l’intestin ont pour conséquence une perte osseuse due à une augmentation du nombre et de l'activité des ostéoclastes, les cellules responsables de la résorption osseuse. Les cellules CD4+ ont été décrites comme participant à la différenciation des ostéoclastes mais la nature exacte de ces cellules reste encore indéterminée. Des études in vivo et in vitro chez la souris et chez l’Homme nous ont permis de décrire une population inflammatoire Th17 TNFα CD4+ présente dans la moelle osseuse participant à la différenciation des ostéoclastes. Ces cellules Th17 participent au recrutement et à la différenciation des monocytes en ostéoclastes en agissant sur les cellules stromales mésenchymateuses. De plus, les maladies inflammatoires chroniques de l’intestin sont associées à une augmentation de la prolifération et de la différenciation des cellules souches hématopoïétiques en cellules myéloïdes. Suite à un stress chimique ou mécanique, les ostéoclastes participent à la mobilisation des cellules souches hématopoïétiques depuis la moelle osseuse. Nous avons montré qu’au cours des maladies inflammatoires de l’intestin, l’augmentation de l’activité des ostéoclastes participe à la prolifération et la différenciation des cellules souches en cellules myéloïdes. Ainsi, les ostéoclastes sont devenus une nouvelle cible thérapeutique intéressante dans la lutte des maladies inflammatoires chroniques de l’intestin / Chronic inflammatory diseases are associated to maintain of memory response and the presence of memory CD4+ T cells. Inflammatory bowel diseases are associated with bone loss due to an increase of the number and activity of osteoclasts, the bone-resorbing cells. CD4+ T cells participated to osteoclasts differentiation, but their nature is still undetermined. In vivo and in vivo studies in mice and human allow us to describe an inflammatory Th17 TNFα CD4+ T cells in the bone marrow that participated to osteoclast differentiation and link inflammation and bone loss. Th17 T cells induce the recruitment and the differentiation of monocytes into osteoclasts acting on mesenchymal stromal cells. Moreover, inflammatory bowel diseases are associated in an increase of hematopoietic stem cell (HSC) mobilization. In stress condition, osteoclasts act on HSC niches and induce their mobilization. We have shown that in inflammatory bowel disease, increase osteoclast activity is involved in the proliferation and differentiation of stem cells into myeloid cells. These myeloid cells are responsible for the intestinal inflammation. Thus, osteoclasts have become an exciting new therapeutic target in the fight of inflammatory bowel diseases

The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the U.S. population over the age of 50. In a similar reach but different clinical realm, nearly 40% of all men and women will be diagnosed with cancer at some point during their lifetimes. The impact of both of these diseases is compounded by the limited knowledge of cellular mechanisms and the insufficiency of effective treatment options. At the microscopic level of the cell cytoskeleton, increasing evidence has led researchers to further explore microtubules (MTs) and MT-based structures, such as primary cilia, as potential keys to unlocking improved treatment options. However, the way in which microtubules regulate the processes giving rise to these diseases remains a critical gap in knowledge. The works outlined here aimed to elucidate mechanisms that may be used to combat diseases attacking the skeletal and immune systems. In order to characterize the influence of primary cilia with respect to osteoclast differentiation, we implemented a series of treatments to an immortalized macrophage cell line: cilia lengthening (using Fenoldopam) and mechanical stimulation (using oscillatory fluid flow). The results were analyzed by a combination of immunocytochemistry and quantitative PCR. Our first result showed definitively that while osteoclasts do not possess primary cilia, their macrophage precursors do. We also discovered that these macrophage primary cilia are dynamic and can be modulated; cells whose cilia had been lengthened showed a significant decrease in osteoclast formation, indicating that macrophage cilia resorption may be a necessary step for osteoclast differentiation to occur. Combined with findings from previous studies, there is increasing evidence that the primary cilium, as a therapeutic target for bone diseases, may offer a dual beneficial approach to both promote bone formation and downregulate osteoclast activity. We then explored the possibility of directional MT translocation during T-cell activation being linked to Rho GTPases, which regulate actin polymerization. WASp and WAVE2, known to have functional roles in T-cell activation, were identified as primary candidates. In order to investigate this relationship, we implemented a stepwise micropatterning procedure by which PDMS was used to transfer local areas of activation (presenting fluorescently-tagged antibodies against CD3 and CD28) which, upon T-cell receptor (TCR) triggering, could mimic immune synapse (IS) formation. We showed that, although there was no correlation between the spatial organization of MTs and WASp, MTs and WAVE2 location were highly correlated, providing strong evidence for a link between these two systems. In addition, MT disruption via nocodazole resulted in a significant decrease in T-cell activation and mechanosensing capabilities. Given the role of WAVE2 in promoting cell spreading and adhesion during IS formation, this result provides additional evidence that this cytoskeletal filament is in fact connected to proteins involved in actin nucleation and elongation. We anticipate the work in Aim 1 to help reveal a previously unexplored therapeutic target for osteoporosis, a disease that currently has no clinical manifestations prior to a fracture event. Further investigation has the potential to contribute to diagnosis and prevention techniques, as well as new treatments. Similarly, given the emergence of adoptive T-cell immunotherapy for immune-related disorders, the findings of Aim 2 will advance our understanding of both the biological and mechanical influence of the cytoskeleton and motivate microtubules as one component of a more comprehensive armamentarium of treatment approaches.

Astronauts are at risk of losing 1.0% to 1.5% of their bone mass for every month they spend in space despite their adherence to diets and exercise regimens designed to protect their musculoskeletal systems. This loss is the result of microgravity-related impairment of osteocyte and osteoblast function and the consequent upregulation of osteoclast-mediated bone resorption. This review describes the ontogeny of osteoclast hematopoietic stem cells and the contributions macrophage colony stimulating factor, receptor activator of the nuclear factor-kappa B ligand, and the calcineurin pathways make in osteoclast differentiation and provides details of bone formation, the osteoclast cytoskeleton, the immune regulation of osteoclasts, and osteoclast mechanotransduction on Earth, in space, and under conditions of simulated microgravity. The article discusses the need to better understand how osteoclasts are able to function in zero gravity and reviews current and prospective therapies that may be used to treat osteoclast-mediated bone disease.

Bone mineral density (BMD) is a reflection of the action of osteoblasts compared to osteoclasts. An imbalance in the activity of osteoblasts or osteoclasts, results in bone disease such as osteoporosis caused by overactive osteoclasts. BMD is influenced by genetic and environmental factors as demonstrated through twin studies, association studies and linkage analysis (Ralston, 1999). Several polymorphisms involved in the determination of BMD have been identified, with Vitamin D receptor and Collagen Type 1 showing reproducible associations. To identify genes influencing BMD two distinct strategies have been employed: 1) To determine if DNA polymorphism within the runt related transcription factor (RUNX2) gene is a determinant of BMD and fracture in women. 2) The identification of RANKL target genes in osteoclastogenesis. RUNX2 is a runt domain transcription factor (Werner et al., 1999) essential for osteoblast differentiation (Lee et al., 1997). RUNX2 gene knock-out mice have no osteoblasts due to a failure in osteoblast differentiation and consequently unmineralised skeletons, (Komori et al., 1997; Otto et al., 1997). In humans, mutations in RUNX2 cause cleidocranial dysplasia (CCD), a disorder characterised by hypoplasia or aplasia of the clavicles, short stature, supernumerary teeth, patent fontanelles and other changes in skeletal patterning and growth (Mundlos et al., 1997). RUNX2 contains a poly-glutamine poly-alanine (polyQ/polyA) repeat where mutations causing cleidocranial dysplasia have been observed. BMD has not been routinely examined in CCD, two studies have identified CCD patients with lower BMD with one fracture case identified (Quack et al., 1999; Bergwitz et al., 2001). The central role of RUNX2 in determining osteoblast differentiation makes RUNX2 a prime candidate gene for regulating adult bone density. To determine if polymorphism was present in the polyQ/polyA tract the repeat was amplified within the upper and lower deciles of femoral neck (FN) BMD in the Geelong Osteoporosis study (GOS). The upper and lower deciles of FN BMD acted as a surrogate for genotyping the entire cohort. This study identified two common variants within the polyA repeat: an 18 base pair deletion (11Ala) and a synonymous alanine codon polymorphism with alleles, GCA and GCG (noted as A and G alleles, respectively). The 11Ala and SNP polymorphism are found on codon 64 and 66 respectively (RUNX2 MRIPV variant). A allele frequencies were significantly different in a comparison of the upper and lower deciles of FN BMD (p=0.019). In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The association was maximal at the ultra-distal radius (p=0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The 11Ala polymorphism was not related to BMD in GOS. To further decipher the role of the RUNX2 A allele we genotyped 992 women from a Scottish cohort. The alleles of RUNX2 within the glutamine/alanine repeat were determined by MspA1I restriction digest. To examine the possible influence on estrogen related therapies or estrogen status on the potential genetic effect conferred by RUNX2, we divided the cohort by menopausal and hormone replacement therapy status. Within postmenopausal Scottish women the RUNX2 A allele was associated with significantly higher FN BMD (p=0.028, n=312) but not lumbar spine (LS) BMD. The A allele was associated with higher FN BMD (p=0.035) within a postmenopausal subgroup of the population (n=312). To investigate the effect of weight on the RUNX2 alleles the Scottish cohort was segregated into thin/normal (BMI ≥ 25 kg/m2) and overweight /obese (BMI > 25 kg/m2). RUNX2 A allele showed a stronger effect on FN BMD in postmenopausal women above the median BMI. The 11Ala RUNX2 deletion allele was significantly associated with decreased LS BMD (p=0.018) within overweight/obese women (n=546). The 11Ala allele was significantly associated with increased levels of pyridinoline (p=0.014) and deoxypyridinoline (p=0.038) in the HRT treated subgroup of the population (n=492). Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 11Ala and A alleles exert differing affects on BMD showing preference for different skeletal sites in a weight dependent manner. We genotyped 78 individuals from an osteoarthritic population to elucidate the role of the RUNX2 alleles on markers of bone turnover and inflammation. The RUNX2 11Ala allele was significantly associated with decreased osteocalcin (OC) serum levels (p = 0.01). The RUNX2 A allele was significantly related to reduced tumor necrosis factor alpha (TNF-alpha) serum levels (p = 0.004). RUNX2 is known to bind to the OC promoter. An OC promoter polymorphism is found 7bp upstream from a putative RUNX2 binding site. We hypothesized that OC polymorphism may effect the RUNX2 transactivation of the OC gene and thus affect OC serum levels. OC promoter polymorphism was not related to OC serum levels (n=78). These data present a novel link between RUNX2 alleles and OC and TNF serum levels, providing putative mechanisms of action for the RUNX2 alleles. Further studies in larger populations are required to confirm these findings. Ten individuals within the GOS and the Scottish cohort were found to carry rare mutations of the polyQ/polyA repeat. All polyQ variants had a normal polyA repeat (17 amino acids) and were heterozygous for a normal 23Q/17A allele. Variants observed were 15, 16, 24 and 30Q. One individual was observed with an extended polyA repeat (24A). Patient records indicated otherwise unremarkable clinical history except for fracture in 4/10 individuals from GOS (hip and spine). BMD data from the LS and the FN were expressed as T-scores, a measure that relates BMD in terms of standard deviations below the young normal value. In addition, BMD data were also expressed as Z-scores around the age-mean. Under the null hypothesis, where RUNX2 Q repeat variation has no effect on BMD, Z scores would be expected to be distributed around a mean of zero. However, when all variants were pooled the BMD was significantly lower than expected. This effect persisted when deletion variants were considered alone. The effect was stronger on FN BMD (p=0.001) rather than LS BMD (p=0.096), reflecting either difference in precision of BMD measurements at these sites or perhaps a differential genetic effect on different skeletal sites. These data suggest that polyQ and polyA variants are associated with significantly lower BMD, and may be an important determinant for fracture. Glutamine variants exist at high frequency (~0.7%): this rate of mutation could be important when considering large populations at risk of age related osteoporosis. Considering that these subjects are heterozygous for a normal allele, it suggests that a more severe phenotype might be expected in rare subjects homozygous for glutamine repeat variants. In summary, this study investigated the role of novel polymorphisms and rare variants of the RUNX2 gene in influencing BMD, fracture and markers of bone turnover. Two common polymorphisms were identified within the polyA repeat: an 18 base pair deletion and a synonymous alanine codon polymorphism with alleles, A and G. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture within a Geelong population. To verify these findings the RUNX2 alleles were genotyped in 992 women from a Scottish cohort. The magnitude and the direction of the effect of the A allele was maintained in the Scottish cohort. Interestingly, the A allele was shown to exert a menopause specific effect, with postmenopausal women showing the strongest effect. On re-analysis of the GOS data the post-menopausal women were found to drive the significance identified in the cohort. The magnitude of the effect of the A allele on BMD was greater in overweight/obese postmenopausal women indicating a gene-weight interaction for RUNX2. The RUNX2 11Ala allele showed a significant relationship with decreased LS BMD in overweight/obese Scottish women. The 11Ala allele was also associated with higher levels of urinary PYD and DPD in women treated with HRT, indicating higher levels of bone turnover in carriers of the 11Ala allele. In contrast to the Scottish cohort, no significant association with heterozygous carriers of 11Ala was observed in GOS, although a significant association was detected for homozygous carriers and LS BMAD. The 11 Ala RUNX2 allele was significantly associated with decreased serum osteocalcin levels and the A allele was significantly associated with TNF in OA patients. Glutamine variants and an alanine insertion were identified within Geelong and Scottish cohorts, which showed low Z and T scores suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. Polymorphism of the polyQ/polyA region of RUNX2 were identified within this study were shown to associate with significant differences in BMD. The A allele showed a significant association with increased BMD in postmenopausal women from a Geelong and Scottish cohort, with a decreased frequency of the A allele observed in Colles' fracture patients from Geelong. The 11Ala deletion allele was significantly associated with decreased LS BMD and increases in markers of bone turnover in the Scottish cohort. A significant decrease in OC serum levels was observed in OA patients suggesting a direct effect of the allele on the transactivation of the RUNX2 gene. Rare variants of RUNX2 were identified which showed low BMD. These studies have provided insight into the role of RUNX2 in influencing BMD, further studies are required to verify the role of the A allele on BMD and fracture, the role of the rare variants and to identify the precise mechanisms behind the observed changes in BMD. - 2) The identification of RANKL target genes in osteoclastogenesis. Osteoclastogenesis is regulated in vivo by the action of osteoblast/stromal cells that express membrane bound, receptor activator of NF-kB ligand (RANKL). Monocytes treated in vitro with a soluble form of RANKL and macrophage colony stimulating factor (M-CSF) differentiate to osteoclasts, whereas monocytes treated with M-CSF alone differentiate to macrophage-like cells. The gene expression profile of human osteoclasts has not been extensively explored. Genes highly expressed by rabbit osteoclasts were identified through random sequencing of an osteoclast cDNA library (Sakai et al., 1995). Differential gene expression of mouse osteoclastogenesis was elucidated by array analysis (Cappellen et al., 2002). To identify genes important for human osteoclastogenesis, total RNA was isolated from monocytes treated for three weeks with either M-CSF alone or with RANKL and M-CSF. RANKL treatment for 3 weeks and 12 hours was investigated in this study, to complement previous data. Differential display was performed on RNA (12 hour treatment with RANKL) and differential gene expression profiles examined. The differential display products were pooled to generate a probe for screening a gene array system derived from a human osteoclast cDNA library. cDNA (3 week treatment with RANKL) hybridisation experiments against the array revealed additional regulated genes. Gene clones that showed significant regulation in M-CSF and RANKL treated cells compared M-CSF treated cells represent genes that are targets for RANKL-specific regulation. Osteopontin, creatine kinase and various mitochondrial genes were up regulated by the treatment of RANKL. Changes in gene expression observed in the array data were confirmed with real-time PCR using mRNA derived from in vitro induced osteoclasts. Cathepsin K gene expression was more than 300 fold greater in osteoclasts compared to macrophage-like cells after one week treatment with RANKL and M-CSF. Cystatin C expression showed a six-fold induction at two weeks of RANKL and M-CSF treatment and cystatin B showed a steady increase in expression. Some of these regulated genes may provide useful targets for influencing BMD.

Osteoporosis, a classical age-related disease and known to be more common in women than in men, has been reported increasingly often in men during the past few years. Although men at all ages after puberty have larger bones than women, resulting in greater bending strength, mortality after a hip fracture, one of the major complications of osteoporosis, is more common in men than in women. Sex hormone deficiency is associated with unrestrained osteoclast activity and bone loss. Even though estrogen deficiency is more pronounced in women, it appears to be a major factor in the pathogenesis of osteoporosis in both genders. In contrast to osteoporosis in postmenopausal women, the treatment of osteoporosis in men has been scarcely reported. Nevertheless, some drugs commonly used for the treatment of osteoporosis in women also appear to be effective in men. The aim of this study is to review primary osteoporosis in the elderly with particular emphasis on gender-related aspects. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.