This is an investigation of the relationships between robusticity and rugosity in the postcranial and cranial skeleton of modern humans. Robusticity is defined here as the strength of an element relative to its size, and refers to the thickness of limb bones for their length, and the relative size of cranial features. Rugosity refers to the surface features of bone, in particular fibrous and fibrocartilaginous entheses. Both data types may provide information about the lives of the individuals to whom the skeletal remains belonged. The two data types may also reflect different remodelling processes in the skeleton. Demographic and environmental variables are investigated in their influence on global variation in both robusticity and rugosity. Subsistence strategy and sex are shown to be the most significant influences on robusticity and rugosity, once other variables are held constant. Body shape and size also influence both cranial and postcranial size and robusticity. However, both robusticity and rugosity demonstrate considerable individual variation both between and within populations. The combination of robusticity and rugosity data in the same analyses is novel, and provides a chance to establish whether the two kinds of data reflect the same underlying osteogenic processes. Robusticity and rugosity in the cranial and postcranial skeleton are shown to correlate only in general terms. This demonstrates that despite the two data types being collected from the same skeletons, they do indeed reflect subtle differences in human skeletal response to demographic, ethnic and environmental influences.

This thesis investigates the size/shape variation in the lumbar spine of extant and fossil hominoids. As a novelty, 3D coordinate data sets were obtained from the last five consecutive presacral vertebrae for comparative analyses. Size/shape variation of single vertebrae and patterns of metameric size/shape variation along the lumbar spine are investigated. Large samples of populations of Homo sapiens, Gorilla gorilla, Pan troglodytes, and Pongo pygmaeus are investigated. The fossil sample includes Australopithecus afarensis, Australopithecus africanus, and Homo ergaster. Statistical shape analysis was conducted using geometric morphometric methods. Comparison 1 (Chapter III) explores sexual dimorphism in size and shape within each of the modern taxa. Results indicate that Pan shows neither vertebral size nor shape dimorphism. Gorilla, and probably Pongo (small sample size) are highly sexually dimorphic in size and less so in shape. Homo sapiens shows less size dimorphism than Gorilla but a markedly larger shape dimorphism. Thus despite sexual dimorphism of locomotor repertoires within great apes there are few indications of these in vertebral shape. In contrast, humans with no dimorphism in locomotor repertoire, show shape dimorphism in the lumbar spine related to sexual differences in pelvic shape and consequent differences in bipedal kinematics. Comparison 2 (Chapter IV) investigates inter-specific size/shape variations between extant hominoids. It is found that differences in shape between the taxa corroborate the functional relationships already described in the literature. Further, the differences between the taxa in shape are not congruent with the consensus molecular phylogeny. Comparison 3 (Chapter V) focuses on the fossils. Results indicate that A. africanus and A. afarensis lumbar vertebrae are most similar to each other. In comparison with modern taxa, they are most similar in shape to those of modern humans and less so to great apes. Homo ergaster falls within the range of modern humans. This thesis concludes that lumbar vertebral morphology shows interesting intra-specific patterns of scaling and of sexual dimorphism that appear to vary according to function between apes and between apes and humans. The australopithecines show similarities in shape with modern humans, indicating that despite inter-specific differences in pelvic shape, there are key adaptations in the lumbar spine which guarantee an energetically efficient bipedalism that was developed as early as 3 million years ago in the hominin lineage. However, humans and australopithecines differ in that the latter show no adaptations in the vertebral column to bipedal endurance running. Chapter VI concludes with a protocol for the analysis of future fossil vertebral discoveries.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Is an endocrine disrupting environmental pollutant that affects bone tissue, although the mechanistic basis of such effect is far from clear. In this study a proteomic approach has been adopted to Investigate the disturbance of the osteogenic process evoked by TCDD In an in vitro osteoblast differentiation model of rat mesenchymal stem cells. Stem cells were isolated from bone marrow of femurs and tibias of rats. Progress of osteoblastic differentiation was monitored by measuring mRNA expression levels of differentiation markers from control and TCDD-treated cells after 3,7 and 10 days of culture In presence and absence of TCDD using quantitative RT-PCR.

Περίπου το 60-70 % της οστεϊκής μάζας αποτελείται από ανόργανα μεταλλικά άλατα και το υπόλοιπο είναι οργανικό υπόστρωμα και νερό. Το κύριο ανόργανο συστατικό είναι ένα μη-στοιχειομετρικό ανάλογο του υδροξυαπατίτη, Ca10(PO4)6(OH)2, (BioHAp) o οποίος απαντάται στην φύση και η οργανική φάση είναι κυρίως (90%) κολλαγόνο τύπου I (COL) ενώ το υπόλοιπο 10 % απαρτίζεται από μια ποικιλία ενώσεων όπως γλυκοπρωτεΐνες, λιπίδια και ένζυμα. Η μελέτη των οστών ως προς της χημική τους σύσταση μπορεί να δώσει χρήσιμα στοιχεία σχετικά με τις ασθένειες των οστών όπως οστεοπόρωση, οστεοπέτρωση, οστεομαλακία, κλπ. αλλά και να βοηθήσει στην αξιόπιστη διάγνωσή τους. Παραδοσιακές αναλυτικές τεχνικές έχουν χρησιμοποιηθεί για την ανάλυση της χημικής σύστασης των οστών, όπως είναι η ηλεκτρονική μικροσκοπία, η περιθλασιμετρία ακτίνων Χ αλλά και η κλασική χημική ανάλυση. Η χρήση αυτών των τεχνικών έχει αποδειχτεί χρονοβόρα, δύσχρηστη και επιπλέον τις περισσότερες φορές είναι καταστρεπτικές για το δείγμα. Η ιδανική αναλυτική τεχνική θα ήταν αυτή που θα απαιτούσε την ελάχιστη προετοιμασία των δειγμάτων χωρίς να υπάρχουν απώλειες στην ποιότητα των πληροφοριών που συλλέγονται από αυτή. Γι’ αυτούς τους λόγους, η φασματοσκοπία Raman θεωρείται ένα πολύ χρήσιμο εργαλείο για τη «χαρτογράφηση» και μελέτη της χημικής σύστασης των οστών. Στην παρούσα εργασία διερευνήθηκε αν η φασματοσκοπία Raman, μια μη καταστροφική δονητική τεχνική με δυνατότητα για σημείο-προς-σημείο ανάλυση ενός υλικού, μπορεί να χρησιμοποιηθεί για αυτό το σκοπό. Από κνημιαία βοοειδή οστά λήφθηκαν φάσματα Raman από το συμπαγές (cortical) και από το σπογγώδες (trabecular) τμήμα του οστού. Για τη μελέτη χρησιμοποιήθηκαν οι χαρακτηριστικές Raman ενεργές δονήσεις στα 960 cm-1 (ν1 PO4 3-) και 2939 cm-1 ν(CH2) για το βιοαπατίτη (BioHAp) και το κολλαγόνο (Col), αντίστοιχα. Τα φάσματα από το συμπαγές και το σπογγώδες τμήμα δεν ήταν άμεσα συγκρίσιμα λόγω της ισχυρής παρουσίας φωσφολιπιδίων και άλλων ουσιών του μυελού των οστών οι οποίες βρίσκονταν στο δικτυωτό τμήμα του οστού. Αναπτύχθηκε πρωτόκολλο απομάκρυνσής τους χωρίς να αλλοιωθεί χημικά το κολλάγονο ή ο βιο-απατίτης, το οποίο διαπιστώθηκε με τηνανάπτυξη πρωτοκόλλων απομόνωσης του βιο-απατίτη και του κολλαγόνου αντίστοιχα. Μετά τον καθαρισμό, λήφθηκε ξανά το φάσμα Raman και χρησιμοποιώντας τους λόγους των δονήσεων BioHAp/Col βρέθηκε ότι ο βιο-απατίτης ήταν περισσότερος στο συμπαγές τμήμα από ότι στο σπογγώδες σε σχέση με το κολλαγόνο. Για την επιβεβαίωση των παραπάνω αποτελεσμάτων μετρήθηκε το ασβέστιο και τα φωσφορικά στα ίδια δείγματα χρησιμοποιώντας τη φασματοσκοπία ατομικής απορρόφησης με χρήση της μεθόδου των σταθερών προσθηκών και τη φασματοφωτομετρία υπεριώδους-ορατού, αντίστοιχα. Τα αποτελέσματα από τις δύο παραπάνω τεχνικές επιβεβαίωσαν ότι το ποσοστό βιο-απατίτη στα συμπαγή οστά είναι μεγαλύτερο σε σχέση με τα σπογγώδη. / Bone is a composite formed by the mineralization of an organic matrix (largely collagen) by the nucleation and growth of a mineral highly resembling calcium hydroxyapatite, Ca10(PO4)6(OH)2, (BioHAp) within the matrix. Seventy percent of mature bone is consisted of the inorganic phase while the organic matrix and water accomplish the rest. The organic phase is predominantly (90%) composed of collagen type I (Col) while a range of other substances such as glycosaminoglycans, glycoproteins, lipids, peptides and enzymes, contribute only to the remaining 10%. Knowledge of the BioHap/Col ratio can permit reliable diagnosis of bone diseases such as osteoporosis, osteopetrosis and osteomalacia. Traditional tools used in assessing the chemical composition of bone include electron microscopy, X-ray diffraction and wet chemical analysis. Usage of such techniques has been proved time-consuming and cumbersome. Furthermore, during the application of these techniques the tissue is exposed to stresses that alter its structure and/or composition. The ideal analytical tool would be one in which minimal tissue preparation is required, whilst allowing no loss of the amount and quality of information derived from the technique. For these reasons the technique of Fourier transform Raman spectroscopy was utilized. In this work Raman spectroscopy, a nondestructive vibrational technique, which permits point-by-point analysis (“mapping”) of a specimen, was applied as a tool for bone chemical analysis. Raman spectra of the cortical and trabecular part of shinbones were recorded. The characteristic Raman vibrations at 960 (v1 PO4 3-) and 2939 cm-1 v(CH2) for BioHAp and collagen, respectively, were used. In order to avoid overlapping of the Raman bands before recording the spectra a sequestration protocol for cleaning the bones from the lipids and the other substances of bone marrow was developed and applied. In order to testify it, two more protocols of isolation bio-apatite and collagen were developed and applied respectively. It was found that the ratio of the Raman intensities BioHAp/Col for the trabecular bone to be lower than the cortical one. For verification purposes the concentrations of calcium and phosphates in the same samples were determined using atomic absorption spectroscopy and the UV-Vis spectroscopy, respectively. It was found that indeed the percentage of bio-apatite in cortical bones is larger than in the trabecular bones.

Bone remodelling is a complex process, which involves the coupling of bone formation to completed foci of bone resorption, the balance between these 2 processes determines if bone is lost or gained at a particular site. During bone resorption osteoclasts release growth factors sequestered in bone matrix, which are thought to initiate new bone formation. On the other hand, osteoblasts can regulate osteoclast activity through the expression of the counter-acting cytokines, RANKL and OPG. The aim of this project was to determine if factors released during bone resorption impact on the RANKL/OPG system or on osteoclasts directly to regulate bone remodelling. OPG secretion was characterized in a number of osteoblast-like cells and the osteosarcoma cell line MG-63 was chosen as a model for osteoblastic cell behaviour in vitro. EDTA bone extracts prepared from normal human cortical bone powder were used to treat MG-63 cells in vitro. The response to the extract was dependent on the purification procedure used. OPG production was inhibited by partially purified extracts prepared using hydrophobic interaction chromatography, C18 SPE. In comparison extracts prepared using size exclusion centrifugal filters stimulated OPG secretion in confluent MG-63 cells. Therefore bone matrix constituents were able to influence osteoclast activity directly and indirectly through the osteoblastic cells to produce the same response. The simplest mechanism for this co-ordinated response would be the presence of one factor in the extract that is able to influence both osteoblasts and osteoclasts. The identity of the factor responsible for the opposing effects seen in the bone matrix extracts is at the moment unknown. The work presented in this thesis clearly demonstrated that unknown growth factors present in bone matrix influence bone remodelling.

L'importance de l'os sous-chondral dans la pathogenèse et la prise en charge de l'arthrose intéresse les cliniciens et la communauté scientifique. En effet, il existe des liens forts entre l'os sous-chondral et le cartilage, maintenant l'intégrité de ce dernier reposant sur l'os sous-chondral pour fournir un support mécanique et un soutien nutritionnel. Ici, nous avons étudié la relation entre les structures osseuses et cartilagineuses et l'approvisionnement vasculaire dans l'arthrose de la humaine.Nous avons recueilli 37 plateaux tibiaux arthrosiques prélevés après arthroplastie totale du genou. Dans ces mêmes plateaux, plusieurs carottes ont été prélevées et scannés par microtomographie. Les projections résultantes ont été reconstruites, puis segmentées manuellement pour séparer l'os sous-chondral de l'os trabéculaire et une analyse microarchitecturale a été développée sous les deux structures osseuses. Les échantillons ont été décalcifiés, coupés en sections de 16 heures, colorés dans de l'HES et classés en 6 groupes selon l'échelle OARSI. La surface de l'os sous-chondral et l'épaisseur et la surface du cartilage articulaire ont été cultivées. Le nombre de vaisseaux dans le sous-chondral a été compté par deux opérateurs différents et une coloration immunofluorescente avec du VEGF a été effectuée. Enfin, le cartilage, l'os sous-chondral et trabéculaire ont été utilisés pour mesurer les marqueurs ribonucléiques et protéiques liés à la vascularisation, l'innervation et l'inflammation.La microstructure de l'os a évolué au fur et à mesure que l'arthrose s'aggrave. L'os sous-chondral s'est épaissi et est devenu plus poreux. La fraction volumique osseuse, l'épaisseur trabéculaire, l'espacement et le nombre de trabécules ont été corrélés positivement avec le score OARSI. Une diminution significative du nombre de vaisseaux sanguins a été observée au dernier stade de l'arthrose. Enfin, les marqueurs ribonucléiques et protéiques liés à la vascularisation, à l'innervation et à l'inflammation ont été modulés au cours du développement de la pathologie. Pris ensemble, nos données montrent une interaction et des structures de soutien dynamiques entre l'os sous-chondral et le cartilage. La compréhension des voies de signalisation, l'unité biochimique du cartilage dans les articulations et la communication intercellulaire entre le cartilage et l'os sous-chondral peuvent mener à l'élaboration de stratégies plus efficaces pour traiter les patients souffrant d'arthrose. / The importance of subchondral bone in the pathogenesis and management of osteoarthritis retain the interest of clinicians and the scientific community. Indeed, there are strong links between the subchondral bone and the cartilage, maintaining the integrity of the latter resting on the subchondral bone to provide mechanical and nutritional support. Here, we investigated the relationship between bone and cartilage structures and vascular supply in human osteoarthritis.We collected 37 osteoarthritic tibial plates taken after total knee arthroplasty. In these same plates, several carrots were removed and scanned by microtomography. The resulting projections were reconstructed, then manually segmented to separate the subchondral bone from the trabecular bone and a microarchitectural analysis was done on both bone structures. The samples were decalcified, cut into 4 μm sections, stained in HES and classified into 6 groups according to the OARSI scale. The surface of the subchondral bone and the thickness and surface of the articular cartilage were measured. The number of vessels in the subchondral region was counted by two different operators and a VEGF immunofluorescent staining was performed. Finally, cartilage, subchondral and trabecular bone were used to measure ribonucleic and protein markers related to vascularization, innervation and inflammation.The microconstructure of the bone has evolved as osteoarthritis worsens. The subchondral bone has thickened and become more porous. Bone volume fraction, trabecular thickness, spacing and number of trabeculae were positively correlated with the OARSI score. A significant decrease in the number of blood vessels was observed in the last stage of osteoarthritis. Finally, ribonucleic and protein markers related to vascularization, innervation and inflammation were modulated during the development of the pathology. Taken together, our data show dynamic interaction and support structures between subchondral bone and cartilage. Understanding of signaling pathways, the biochemical unity of cartilage in the joints and intercellular communication between cartilage and subchondral bone can lead to the development of more effective strategies for treating patients with osteoarthritis.

Le rôle de la microstructure trabéculaire et corticale dans la résistance osseuse et le risque de fracture est bien documenté mais son déterminisme génétique n’a pas encore été étudié. Pour savoir si la microarchitecture osseuse et le métabolisme osseux ont un déterminisme héréditaire, une étude épidémiologique transversale (étude MODAM) a été menée pour évaluer la ressemblance familiale de la microarchitecture osseuse chez les femmes ménopausées et leurs filles non ménopausées en utilisant la tomodensitométrie quantitative périphérique haute résolution (HR-pQCT). Nous avons constaté que les filles des femmes ayant subi une fracture ont une densité osseuse volumétrique totale (vBMD) plus faible, des corticales amincies, et une altération de la microarchitecture osseuse trabéculaire au niveau du radius distal et du tibia comparativement aux filles dont les mères n’ont pas eu de fracture. Une autre étude épidémiologique transversale (étude MINOS) a été menée pour évaluer la corrélation de la densité minérale osseuse (DMO), de la taille des os, des marqueurs du remodelage osseux (BTM) et des hormones de régulation du remodelage osseux dans des paires de frères et des paires d'hommes non apparentés, appariés pour l'âge, le poids et la taille. Nous avons constaté que la taille des os, la DMO à certains sites du squelette et la plupart des BTM étaient plus fortement corrélés chez les frères comparativement aux hommes non apparentés. Ces données suggèrent un déterminisme héréditaire substantiel des niveaux de BTM chez les hommes. En conclusion, nous développons et confirmons l'importance des facteurs familiaux dans la pathogénie de l'ostéoporose chez les femmes et les hommes / The role of trabecular and cortical microstructure in bone strength and fracture risk is well documented, but its genetic determinism has not yet been studied. To find whether the bone microarchitecture, and bone metabolism have a strong hereditary determinism, a crosssectional epidemiological study (MODAM study) was conducted, investigating the familial resemblance of bone microarchitecture in postmenopausal mothers and their premenopausal daughters using high-resolution peripheral quantitative computed tomography (HR-pQCT). We found that daughters of women with fracture have lower total volumetric bone mineral density (vBMD), thinner cortices, and impaired trabecular microarchitecture at the distal radius and tibia, compared with daughters of women without fracture. Another cross-sectional epidemiological study (MINOS study) was conducted to assess the correlation of bone mineral density (BMD), bone size, bone turnover markers (BTMs) and hormones regulating bone turnover in pairs of brothers and pairs of unrelated men, matched for age, weight and height. We found that bone size, BMD at some skeletal sites and most of the BTM levels correlated more strongly in the brothers than in the unrelated men. These data suggest a substantial hereditary determinism of the BTM levels in men. In conclusion, we expand and confirm the importance of familial factors in the pathogenesis of osteoporosis in both women and men

Alors que les phases de résorption et de couplage du cycle de remodelage de l’os sont de plus en plus connues et ont permis le développement d’agents thérapeutiques, la phase d’activation reste peu étudiée. L’objectif global de ce travail est d’analyser les évènements cellulaires mis en jeu au cours de la phase d’activation du remodelage de l’os. Les objectifs spécifiques ont été 1- de caractériser le trafic cellulaire dans le périoste au cours de la phase d’activation du remodelage et 2- d’étudier le rôle d’une enzyme, la nicotinamide phosphorybosyl transférase (Nampt) dans ces évènements. Dans notre premier travail, nous montrons dans un modèle de remodelage synchronisé de l’os alvéolaire, une expression précoce de ICAM-1 par les vaisseaux qui serait impliquée dans la diapédèse observée de monocyte-macrophages CD68+. Ces cellules migreraient à travers le compartiment non ostéogénique puis ostéogénique, guidées par des cellules de type fibroblastes puis des OB exprimant VCAM-1. Le nombre des cellules RANKL+ dans le compartiment ostéogénique augmente graduellement lors de la phase d’activation. En parallèle, l’expression de la sémaphorine 3a, qui inhibe l’ostéoclastogénèse, diminue chez les OB et les ostéocytes superficiels. Dans notre second travail, nous trouvons que l’expression basale de la Nampt est accrue dans les cellules de la couche ostéogénique au cours de la phase d’activation du remodelage. Inhiber son activité via le FK866 permet de diminuer l’ostéoclastogenèse indiquant que la Nampt serait impliquée dans le recrutement et l’activité des OC. En culture primaire d’ostéoblastes murins, nous montrons que son expression augmente au cours de la différentiation et qu’elle régule l’expression de marqueurs tardifs de différentiation. L’ensemble de ces données montre une série d’évènements coordonnés qui servent au recrutement des précurseurs ostéoclastiques et à leur migration vers la surface osseuse à résorber. La Nampt semble jouer un rôle dans l’acquisition des ostéoblastes d’un phénotype favorable à ces évènements. / Resorption and inversion phases of bone remodeling are well understood, which have permitted the development of therapeutic agents. At the opposite, activation phase remains poorly characterized. This work aims to analyze cellular events involved in the activation phase of bone remodeling. Specific goals were: 1- To characterize cellular traffic in the periosteum during the activation phase of bone remodeling. 2- To study the role of NicotinAMide Phosphorybosyl Transférase (NAMPT) enzyme during activation. In the first study, we show an early expression of ICAM-1 by vessels in a synchronized alveolar-bone-remodeling model. The ICAM-1 expression may be involved in the observed diapedesis of monocytes – macrophages CD68+. These cells migrate through non osteogenic and osteogenic layers, steered by fibroblast-like cells and then by VCAM+ osteoblasts (OB). The number of RANKL+ cells in osteogenic layer gradually increases during the activation phase. Simultaneously, the expression of semaphorine 3a inhibiting osteoclastogenesis, decreases in osteoblasts and superficial osteocytes. In the second study, we show that basal expression of NAMPT increases in osteogenic-layer cells during the activation phase of bone remodeling. Inhibiting its activity with FK866 enhables to decrease osteoclastogenesis, suggesting an involvement of NAMPT in osteoclast recruitment and activity. In primary culture of murine OB, we show that NAMPT expression increases during differentiation. It also regulates OB late-differentiation markers expression. All these data show a series of coordinated events which serve in osteoclasts precursors’ recruitment and migration towards bone surface. NAMPT seems to contribute to acquiring an OB phenotype more favorable to OC recruitment.