Mesenchymale Stammzellen können sich durch den Einfluss verschiedener Transkriptionsfaktor zu Osteoblasten, Adipozyten, Chondrozyten oder Myoblasten differenzieren. Während sie sich unter Runx2-Einfluss entlang der osteoblastären Linie differenzieren, entwickeln sie sich bei vorliegendem PPARγ entlang des adipogenen Differenzierungswegs. Das Gleichgewicht zwischen beiden Faktoren und ihr Zusammenspiel stellen einen wichtigen Bereich in der Osteoporoseforschung dar. In dieser Dissertation wurde durch Runx2-Suppression bzw. Runx2-Überexpression die Rolle dieses Faktors in pHOB und SCP1-Zellen erfasst und die Interaktion zwischen Runx2 und PPARγ untersucht. Der Runx2-Knockdown’ erfolgte mittels RNA-Interferenz, die Runx2-Überexpression durch ein Runx2 exprimierendes Plasmid. In RT-PCRs wurden mRNA-Messungen durchgeführt. Die Proteinbestimmung erfolgte im ‚Westernblot’. Der funktionelle Einfluss der Runx2-Überexpression auf die PPARγ-Transkription wurde durch Kotransfektion des an Luziferase gekoppelten PPARg-Promotorgens erfasst. Die funktionelle Aktivität des PPARg-Proteins wurde durch die Transfektion des an Luziferase gekoppelten PPRE-Gens gemessen. Promotoraktivität und Funktionalität der Proteine wurden in Luziferase-Reportergenassays erfasst. Unter basalen Kulturbedingungen differenzierten sich pHOB osteogen. Durch zweimalige siRunx2-Transfektion gelang auf mRNA-Ebene eine suffiziente Runx2-Suppression über 29 Tage auf durchschnittlich 10,1%. Neben einer Steigerung der PPARγ-mRNA nach sieben Tagen konnte darunter auch eine Suppression der osteogenen Differenzierungsmarker OC und AP beobachtet werden. Ein ‚Rescue’ der supprimierten Runx2-Genexpression konnte durch osteogene Stimulation nicht erreicht werden. In den Runx2-/PPARγ-Interaktionsversuchen wurden SCP1-Zellen adipogen stimuliert, um die PPARγ2-mRNA und PPARγ-Promotoraktivität zu erhöhen. Darunter konnte ebenfalls eine gesteigerte Funktionalität des PPARγ-Proteins beobachtet werden. Durch Runx2-Überexpression wurde in SCP1-Zellen die PPARγ-Promotoraktivität und somit der Beginn der mRNA-Synthese gehemmt. Die PPARγ2-mRNA hingegen blieb unbeeinflusst. Die zentrale Rolle des Runx2 in der osteogenen Differenzierung scheint durch den Einfluss auf die osteogenen Marker OC und AP in pHOB bestätigt zu werden. Auch der Einfluss auf die adipogene Differenzierung erfolgt über Runx2. Im Rahmen dieser Dissertation konnte erstmalig die Hemmung des PPARγ-Promotors durch Runx2 beschrieben werden. Hierdurch werden die PPARγ-Transkription und somit voraussichtlich die Interaktion zwischen Adipogenese und Osteogenese beeinflusst.

No description available.

Vitamin D is a precursor to a steroid hormone, 1,25 dihydroxyvitamin D (1,25(OH)2D). After its discovery and the characterization of its receptor, the vitamin D receptor (VDR), it was initially thought only to be involved in calcium homeostasis, but further research revealed an important role for vitamin D in the regulation of cell growth and differentiation of such cells as osteoblasts and bone marrow adipocytes. 1,25(OH)2D has also been shown to be a strong inhibitor and pro-differentiator of keratinocytes. The anti-proliferative and pro-differentiative properties of this hormone have led to studies where 1,25(OH)2D anticancer properties were assessed and initial findings that showed a requirement of other factors beyond VDR to induce 1,25(OH)2D signaling led to the identification of the retinoid X receptor, a common heterodimeric partner for several hormone receptors. The focus of thesis was to further elucidate the structure-function relationship of both the vitamin D receptor and the retinoid X receptor. Additionally, contributions to work directed towards further identifying the effects of vitamin D on osteoblast differentiation and survival. Interactions of 1,25(OH) 2D3 with its cognate receptor, identifying a key amino acid (Tryptophan 286) required for ligand contact and transcriptional activation, are described in Chapter 2. Mechanisms of vitamin D action on mesenchymal stem cell differentiation, promotion of osteoblast induction and maturation, and inhibition of adipocyte differentiation, are eluicidated in Chapter 3. Chapter 4 illustrates the effects of RAS/RAF/Mitogen-activated protein kinase mediated RXRalpha phosphorylation on the three-dimensional structure of the RXR/nuclear receptor partner heterodimers. Furthermore, this chapter reveals the inhibitory effect of the phosphorylation of a critical amino acid (serine 260) on the interaction of the AF-2 domain of the RXR with several coactivators, resulting in a decrease in the signaling potential of multiple steroid hormone receptors. The findings of this thesis further the knowledge of several areas of vitamin D biology, including both the canonical areas of bone formation, and the non-canonical area of vitamin D and cancer.

Distraction osteogenesis (DO) is a surgical procedure in which applied strain stimulates new bone growth; however, the underlying mechanisms by which bone cells respond to load are still uncertain. An organ culture model of DO was developed and validated by using linear distraction on the femoral shafts of 5 day old Wistar rats. Two loading regimes were utilized: distracting the bones for 2 hrs on day 1 (GRP I); distracting the bones for 2 hrs on days 1, 3, and 5 (GRP II). After 1 week in culture, the bones were compared to unloaded contralateral controls and assessed for changes. Structural, dimensional, massing, micro-CT, areal, and viability properties were obtained from testing. Relative to paired controls, distracted bones demonstrated an increase in failure load (9.15% GRP I, 18.85% GRP II), increase in stiffness (31.28% GRP I, 53.21% GRP II), increases in areal and polar moments of inertia, and viability (6.21% GRP I, 13.02% GRP II). Our results suggest that DO can be modeled successfully with an organ culture, and continued use of this system will help to gain insight into the mechanisms and pathways by which distraction osteogenesis occurs.

The objective of the present study was to synthesize porous, biodegradable poly (D, l- lactide-co-glycolide) PLGA-B-HA (Bovine hydroxyapatite) composite and evaluate the effect of ceramic content on bone marrow cell differentiation in vitro. A macroporous biodegradable PLGA-B-HA composite with the pore size varying from 0.1 to 1000?? and a highly interconnected structure was fabricated using the freeze-drying/lyophilization technique. A pilot study was done to determine the effects of B-HA on to the osteoblast function. The main study was done to determine the effect of the increase in B-HA concentration on to the mesenchymal stem cell differentiation. Morphological characteristics of the composites were analyzed using FTIR and SEM/EDX analysis. The composites were seeded with neonatal rat calvarial osteoblasts (NRCO). The polymer: ceramic ratio in this study was 35%:65%. For comparison parallel experiments involving pure HA-200 discs were performed. SEM results indicated a higher proliferation and mineralization on PLGA-B-HA composites than pure HA discs. In addition, we evaluated the in vitro characteristics of PLGA-B-HA composites with varying ratios, i.e., 1:1, 1:2 and 1:3, seeded with rat marrow cells. FTIR indicated an increase in the area under the ceramic peak as ceramic concentration was increased. In addition, the average roughness values increased in the order of 1:3 andgt; 1:2 andgt; 1:1. Both compressive strength and modulus of 1:1 were significantly higher than 1:2 and 1:3 PLGA-B-HA composites. No significant difference in compressive modulli and strengths could be observed for 1:2 and 1:3 PLGA-B-HA composites. Cellular activity was determined by measuring AP activity, total protein analysis and osteocalcin concentration. Evaluation of alkaline phosphatase activity showed bone cells attached to 1:3 (PLGA-B-HA) expressed significantly higher alkaline phosphatase as compared to 1:1 and 1:2 PLGA-B-HA composites. In addition, cells seeded on to 1:3 composites secreted significantly higher osteocalcin and at a relatively short time period as compared to the other samples. Corrosion studies (ICP) and pH values indicate minimal difference in the concentration of Ca and P and pH in tissue culture media for all the samples at the end of all time periods. Hence we conclude that an increase in the ceramic concentration stimulated mesenchymal stem cell differentiation thereby promoting osteogenesis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 18, 2007) Vita. Includes bibliographical references.

Η διπλωματική αυτή εργασία πραγματεύεται τη μελέτη της μεθόδου Ilizarov καθώς και το σχεδιασμό, την κατασκευή και τον έλεγχο ενός ισοδύναμου με ένα βαθμό ελευθερίας του πλαισίου Taylor στοιχείου δράσης, του οποίου η ενεργοποίηση επιτυγχάνεται με τη χρήση έξυπνων υλικών και συγκεκριμένα μεταλλικών κραμάτων με μνήμη της μορφής (SMA). Πρωτεύον μέλημά μας αποτελεί η μελέτη των γενικών χαρακτηριστικών της εξωτερικής σταθεροποίησης και η κατανόηση της μεθόδου Ilizarov με τη χρήση του πλαισίου Taylor (Κεφάλαιο 2). Ακολούθως, κάνουμε λόγο για τους ενεργοποιητές που χρησιμοποιούνται στη διάταξή μας, αναλύοντας εκτενώς τις ιδιότητες των κραμάτων με μνήμη της μορφής και πιο συγκεκριμένα των κραμάτων NiTi (Κεφάλαιο 3). Λαμβάνοντας υπόψη όλα τα προηγούμενα, είμαστε σε θέση να προχωρήσουμε στην υλοποίηση μίας κατασκευής που προσομοιώνει ένα βαθμό ελευθερίας του πλαισίου Taylor και η οποία ενεργοποιείται από τα παραπάνω έξυπνα υλικά. Πειράματα ανοικτού και κλειστού βρόχου ελέγχου της διάταξης εκτελούνται εν συνεχεία με τη βοήθεια ηλεκτρονικού υπολογιστή (Κεφάλαιο 4). Τέλος, παρουσιάζονται τα συμπεράσματα και οι προοπτικές της εν λόγω εργασίας (Κεφάλαιο 5). / The present diplomatic work consists of the study of Ilizarov method as well as the planning, the construction and the control of one out of six degrees of freedom of a Taylor platform. The activation of the experimental provision is achieved with the use of intelligent material and concretely shape memory alloys (SMA). Our first concern is the study of the general characteristics of the exterior stabilization of the bones and the comprehension of Ilizarov method using the Taylor platform (Chapter 2). Followingly, we deal with the activators that are used in our provision, analyzing extensively the attributes of shape memory alloys and more concretely the alloys NiTi (Chapter 3). Taking into consideration all the above, we are in position to advance in the concretisation of a structure that simulates one degree of freedom of the Taylor platform and is activated by the SMA. We executed control experiments using LabView in an open and closed loop in order to test the provision (Chapter 4). Finally, are presented the conclusions and the prospects of this project (Chapter 5.)