O período embrionário apresenta maior susceptibilidade a teratógenos, sendo a fase que as primitivas camadas germinativas e rudimento dos órgãos são formados. Cada órgão tem uma fase crítica de desenvolvimento e os processos bioquímicos envolvidos no crescimento e diferenciação tem uma seqüência ordenada, controlada por vários genes. A ossificação endocondral tem inicio sobre um molde de cartilagem hialina, com formato semelhante ao osso que irá se originar, sendo o principal responsável pela formação dos ossos curtos e longos. Assim, julgamos ser de grande valia um estudo embasado no desenvolvimento normal dos membros de embriões e fetos bovinos provenientes de vacas criadas em ambiente natural, através de estudos em microscopia de luz e microscopia eletrônica de transmissão. No processo de formação, desenvolvimento e ossificação dos membros observamos a formação de tecido mesenquimal condensado em embriões por volta da quarta semana gestacional, passando pelo processo de diferenciação em células cartilaginosas. Ao redor da sétima semana gestacional inicia-se o processo de calcificação da cartilagem com depósitos de cálcio e fósforo para formação de tecido ósseo, seguido de morte dos condrócitos. / The embryonary period presents a bigger susceptibility to theratogens, being the phase in which the primitive germinative layers and rudiment of organs are been formed. Each organ has a critical phase of development and the biochemical processes involved in growing and differentiation have an ordered sequence, controlled by several genes. The endochondrial ossification has begun over a hyaline cartilage mould, having a shape similar to the bone that will be grow, being the principal responsible for the short and long bones formation. Thus, we think it is much valid a study based on normal development of bovine embryos and fetuses\' limbs coming from cows bred in a natural environment, through studies in light microscopy and transmission electronic microscopy. In the process of formation, development and ossification of limbs, we saw condensed mesenchymal tissue formation in the embryos at around the gestational fourth week, passing through differentiation process in cartilaginous cells. At around the gestational seventh week, it has begun the calcification process of cartilage with calcium and phosphorous deposits to the bone tissue formation, followed by chondrocites death.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Cartilage tissue provides compressive resistance in diarthrodial joints, and has been shown to be regulated by mechanical signals, in particular with regard to production of extracellular matrix proteins. However, less is understood about how chondrocytes in regions not solely purposed to provide compressive resistance may also be affected by mechanical forces. The growth plate is a small layer of cartilage that functions to facilitate longitudinal growth of the long bones from in utero through post-adolescent development. The growth plate maintains distinct regions of chondrocytes at carefully regulated stages of endochondral ossification that are in part characterized by their morphology and differential responsiveness to vitamin D metabolites. Understanding if mechanical cues could be harnessed to accelerate or delay the process of endochondral ossification might be beneficial for optimizing tissue engineering of cartilage or osteochondral interfaces. This study focused on three aims to provide a basis for future work in this area: 1) Develop a cell line culture model useful for studying growth plate chondrocytes, 2) Determine the response of primary growth plate chondrocytes and the cell line model to fluid shear stress, and 3) determine if expression of integrin beta 1 is important for the observed responses to shear stress. The findings of this study suggest that inorganic phosphate can promote differentiation in coordination with the 24,25(OH)2D3 metabolite of vitamin D, and that fluid shear stress generally inhibits differentiation and proliferation of growth plate chondrocytes in part through an integrin beta 1 mediated pathway.

It is a well established phenomenon that the epithelial lining of the urinary bladder (urothelium) when implanted into skeletal muscle induces ectopic ossification. However, despite numerous observations, this reaction is poorly understood. This research further studied this reaction by - (a) demonstrating the reaction in a suitable small animal model; (b) attempting to induce the reaction by implanting urothelial cells purified by cell culture techniques; and (c) comparing the bone forming reaction induced by implanted urothelium to the reaction induced by implanting Bone Marrow Stem Cells (BMSC's) and Osteophyte Stem Cells (OSC's). By demonstrating newly formed bone after the implantation of guinea pig urothelium into the skeletal muscle of a Severe Combined Immuno-Deficient Mouse (SCID-Mouse) this research demonstrated that a suitable small animal model had been established. This is despite inherent difficulties (particularly bacterial contamination) associated with establishing a primary cell culture of guinea pig urothelial cells. Additionally, the intramuscular ectopic osteoinductive potential of human BMSC's (hBMSC's) in the SCID-mouse has also been demonstrated. Confirming that the injection of cultured cells in suspension is an adequate intramuscular delivery technique, this research demonstrates that hBMSC's induce ectopic ossification by non-immunological means. This research has demonstrated a number of differences between urothelium induced ectopic ossification and ectopic ossification induced by BMSC's, suggesting they are two separate processes. This is important because the chemotaxis and subsequent osteogenic differentiation of BMSC's has previously been one of the more popular postulated mechanisms of urothelium induced ectopic ossification. Finally, this research has demonstrated the ectopic osteoinductive potential of stem cells isolated from the marrow of human osteophytes (human Osteophyte Stem Cells, hOSC's). This observation has not been previously reported, and will hopefully provide a valuable contribution to a body of knowledge that has important ramifications in both the treatment of osteoarthritis, and the use of BMSC's in tissue engineering.

Thesis (MSc.(Anatomy)--Faculty of Health Sciences)-University of Pretoria, 2007. / Includes bibliographical references.

Η ετερότοπη οστεοποίηση είναι ένα όχι σπάνιο φαινόμενο που οδηγεί σε δημιουργία οστικών δομών σε σημεία που φυσιολογικά υπάρχουν μαλακά μόρια. Ετερότοπη οστεοποίηση μπορεί να προκληθεί κατόπιν τοπικού τραύματος, κατόπιν νευρολογικού τραύματος, ύστερα από χειρουργική επέμβαση σε περιοχές όπως τα ισχία και οι αγκώνες, λόγω γενετικού υποστρώματος σε ασθενείς πολύ μικρών ηλικιών και τέλος αντιδραστικές ετερότοπες οστεοοποιήσεις άνω ή κάτω άκρων. Στην παρούσα διατριβή προσεγγίστηκε η νευρογενούς αιτιολογίας ετερότοπη οστεοποίηση, κυρίως κατόπιν ΚΕΚ. Ο παθοφυσιολογικός μηχανισμός του φαινομένου είναι εν πολλοίς άγνωστος και αυτό που θεωρείται δεδομένο είναι η διαταραχή του ισοζυγίου οστεοβλαστικής – οστεοκλαστικής δραστικότητας κατόπιν της δράσης του επαγωγικού παράγοντα. Ύστερα από τη δράση του επαγωγικού παράγοντα –στη συγκεκριμένη περίπτωση της ΚΕΚ – αυξάνεται η οστεοβλαστική δραστηριότητα τοπικά. Κατά το σχηματισμό του οστού λοιπόν, παράγονται και εκκρίνονται πρωτεογλυκάνες στις αλυσίδες των οποίων προσκολλώνται οι γλυκοζαμινογλυκάνες. Πρωτεογλυκάνες και γλυκοζαμινογλυκάνες συναποτελούν μαζί με τις κολλαγονικές και μη κολλαγονικές πρωτεΐνες, τα τρία κύρια είδη μακρομορίων του εξωκυττάριου δικτύου του οστού. Σκοπός της μελέτης μας ήταν αφενός η μελέτη των πρωτεογλυκανών και γλυκοζαμινογλυκανών στο ετερότοπο οστό σε αντιδιαστολή με φυσιολογικό-ορθότοπο οστό προκειμένου να διερευνηθεί ο ρόλος τους στην δημιουργία του φαινομένου της ετρότοπης οστεοποίησης. Αφετέρου για να διερευνηθεί καλύτερα ο παθοφυσιολογικός μηχανισμός του φαινομένου, μελετήθηκαν κυτταρικοί πληθυσμοί με οστεοβλαστική δραστηριότητα στο περιφερικό αίμα ασθενών που είχαν υποστεί ΚΕΚ και νοσηλεύονταν στη ΜΕΘ, καθώς και πειραματοζώων κατόπιν τεχνητής επαγωγής Κρανιοεγκεφαλικής Βλάβης. Η θειική χονδροϊτίνη και το υαλουρονικό οξύ είναι οι μοναδικοί τύποι γλυκοζαμινογλυκανών στο εξωκυττάριο δίκτυο ετερότοπου οστού όπως και στο φυσιολογικό. Εντούτοις, το ολικό ποσό τους είναι κατά 70% μικρότερο σε σύγκριση με αυτό του φυσιολογικού οστού. Διαφορετική είναι και η εκατοστιαία αναλογία αυτών των μακρομορίων. Η επικρατούσα μορφή δισακχαρίτη θειικής χονδροϊτίνης είναι η θειωμένη στην θέση 6. Ωστόσο η ποσοτική διαφοροποίηση από το φυσιολογικό οστό τόσο στους 4 θειωμένους όσο και στους μη θειωμένους δισακχαρίτες είναι υπαρκτή σε όλα τα ετερότοπα δείγματα. Από πλευράς πρωτεογλυκανών η αγγρικάνη και η διακοσμητίνη είναι ποιοτικά παρούσες στο εξωκυττάριο δίκτυο οστίτη ιστού. Επομένως, ποσοτικές διαφοροποιήσεις στο ετερότοπο οστό σε αντιδιαστολή με το φυσιολογικό είναι υπαρκτές και αυτή η διαφοροποίηση πιθανώς αντικατοπτρίζει διαφορετικές ενζυμικές δραστηριότητες στο φαινόμενο της ετερότοπης οστεοποίησης. Στη μελέτη των κυτταρικών πληθυσμών με οστεοβλαστική δραστηριότητα στο περιφερικό αίμα διαπιστώνονται τα ακόλουθα: Αυξημένη οστεοβλαστική δραστηριότητα στους πληθυσμούς CD-63(+) η οποία εμφανίζει κορυφή στις 6-10 ημέρες μετά την ΚΕΚ. Αυξανόμενη οστεοβλαστική δραστηριότητα πληθυσμών κυττάρων osteocalcin (+) σε όλες τις μετρήσεις μετά την ΚΕΚ. Το σύστημα οστεοπροτεγερίνης – sRANKL εμφανίζει τα εξής χαρακτηριστικά: η osteoprotegerin είναι μετρήσιμη και αυξάνει προς το τέλος των μετρήσεων. Το sRANKL απεναντίας δεν είναι μετρήσιμο σε καμία χρονική στιγμή κατόπιν της ΚΕΚ. Τα παραπάνω συνεπάγονται ότι η ΚΕΚ είναι παράγων επαγωγής οστεοβλαστικής δραστηριότητας όχι μόνο τοπικά αλλά και συστηματικά. Η εκτροπή της οστεοβλαστικής δραστηριότητας προς δημιουργία ετερότοπης οστεοποίησης χρήζει μελέτης μεγαλυτέρου δείγματος ασθενών και πιθανότατα και σε επίπεδα γονιδιακής έκφρασης κυτταρικών καλλιεργειών ασθενών κατόπιν ΚΕΚ. / Ηeterotopic ossification is a relatively frequent phenomenon that leads to the formation of heterotopic osseous structures at points where soft molecules normally do exist. Heterotopic ossification can be induced after local lesion, neurological lesion, after surgical intervention in regions as the hips and the elbows, due to genetic causes in patients of very small ages and, finally, the phenomenon has been observed as distinct, reactive cases in upper or lower limbs. In this Thesis, pathophysiology and mechanisms of neurogenic heterotopic ossification were studied. Pathways of the phenomenon still unknown to date. What is thaught to be the case in the formation of HO, is the disturbance of balance of osteoblastic to osteoclastic activities, after the induction-Head injury in this situation. After Traumatic Brain Injury, osteoblastic activity is induced locally. During bone formation proteoglycans are produced and secreted. Glucozaminoglycans are attached on the side chains of Proteoglycans. Proteoglycans and Glycozaminoglycans constitute along with collageneous and non-collageneous proteins the major macromolecules of extracellular matrix. The purpose of our study was the characterization of proteoglycans and glycozaminoglycans of the heterotopic bone versus the normotopic bone towards the elucidation of their role in the heterotopic bone formation. On the other hand, a more detailed approach to the pathophysiology of the phenomenon requires cellular populations expressing osteoblastic activities to be observed and studied. This is done in peripheral blood of patients that had sustained traumatic brain injuries and being hospitalized within IC units. Same studies on cellular populations have been conducted in a rabbit animal model of traumatic brain injury. Chondroitin-Sulfate and Hyaluronate are the only glycozaminoglycans that have been observed in extracellular matrix of heterotopic bone as well as in normotopic one. Quantitative analyses, however, revealed that their total amount is 70% less compared to normotopic bone. The commonest form of dissacharites of chondroitin-sulfate is the one sulfated at 6-O. However, there is a significant quantitative difference between normotopic and heterotopic bone in 4-O sulfated as well as in non sulfated dissacharites. With regards to proteoglycans, aggrecan and decorin are present in extracellular matrix. Quantitative differences between normotopic and heterotopic bone do exist and reflect a possible alternative pathway of bone formation in the HO phenomenon. The studies on osteoblastic activities of peripheral blood after traumatic brain injury revealed that there is increased osteoblastic activity in CD-63 (+) population that peaks 6-10 days after the inciting event. Osteocalcin (+) population do excibit increased osteoblastic activity as well which increases along with time. The system osteoprotegerin - sRANKL presents the following characteristics: osteoprotegerin is measurable and increases towards the end of measurements. sRANKL on the contrary is not measurable at any time following traumatic brain injury. Consequently, Traumatic Brain Injury do induce osteoblastic activity not only locally but also systemically. The deviation of osteoblastic activity towards heterotopic ossification requires studies of bigger sample of patients, probably to the level of differential gene expression of cellular cultures derived from patients having sustained neurotrauma.

O período embrionário apresenta maior susceptibilidade a teratógenos, sendo a fase que as primitivas camadas germinativas e rudimento dos órgãos são formados. Cada órgão tem uma fase crítica de desenvolvimento e os processos bioquímicos envolvidos no crescimento e diferenciação tem uma seqüência ordenada, controlada por vários genes. A ossificação endocondral tem inicio sobre um molde de cartilagem hialina, com formato semelhante ao osso que irá se originar, sendo o principal responsável pela formação dos ossos curtos e longos. Assim, julgamos ser de grande valia um estudo embasado no desenvolvimento normal dos membros de embriões e fetos bovinos provenientes de vacas criadas em ambiente natural, através de estudos em microscopia de luz e microscopia eletrônica de transmissão. No processo de formação, desenvolvimento e ossificação dos membros observamos a formação de tecido mesenquimal condensado em embriões por volta da quarta semana gestacional, passando pelo processo de diferenciação em células cartilaginosas. Ao redor da sétima semana gestacional inicia-se o processo de calcificação da cartilagem com depósitos de cálcio e fósforo para formação de tecido ósseo, seguido de morte dos condrócitos. / The embryonary period presents a bigger susceptibility to theratogens, being the phase in which the primitive germinative layers and rudiment of organs are been formed. Each organ has a critical phase of development and the biochemical processes involved in growing and differentiation have an ordered sequence, controlled by several genes. The endochondrial ossification has begun over a hyaline cartilage mould, having a shape similar to the bone that will be grow, being the principal responsible for the short and long bones formation. Thus, we think it is much valid a study based on normal development of bovine embryos and fetuses\' limbs coming from cows bred in a natural environment, through studies in light microscopy and transmission electronic microscopy. In the process of formation, development and ossification of limbs, we saw condensed mesenchymal tissue formation in the embryos at around the gestational fourth week, passing through differentiation process in cartilaginous cells. At around the gestational seventh week, it has begun the calcification process of cartilage with calcium and phosphorous deposits to the bone tissue formation, followed by chondrocites death.