Entwicklung und Etablierung eines auf sekundären, murinen Osteoblasten und Fibroblasten beruhenden Co-Kultur-Modells zur genomischen Analyse von Wachstums- und Differenzierungsfaktoren und Evaluierung dieser neuen Methode am Beispiel des Bone Morphogenetic Proteins 7

Die Verletzungen der Kreuzbandstrukturen des Kniegelenkes nehmen eine zentrale Bedeutung in der Orthopädie und Unfallchirurgie ein und weisen aufgrund der hohen Inzidenz eine sozioökonomische Relevanz auf. Eine operative Versorgung der vorderen Kreuzbandruptur wird vor allem bei Instabilitäten des K...

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Bibliographic Details
Main Author: Pado, Michael
Contributors: Lechler, Philipp (Prof. Dr. med.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
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Lesions of the crucial ligaments of the knee joint play an eminent role in the field of orthopaedics and traumatology. Their high incidence makes them highly socio-economically relevant. Surgical treatment of tears of the anterior crucial ligament is required especially when instability occurs. This can prevent possible subsequent damages such as secondary meniscal lesions or posttraumatic gonarthrosis. Despite the continuous optimization of the surgical treatment after tears of the anterior crucial ligament, rerupture rates are still high. These are caused by inadequate surgical technique, insufficient patient compliance and also to failure of the biological integration of the transplant. In this context, complex biological processes (inflammation, reparation, remodelling) of the bone-tendon integration form a central research focus. This can helpt to better understand incorrect or poor transplant incorporation and to ensure a stable and quick bone-tendon-healing. Osteoblasts and fibroblasts play an important role in the process of osseous tendon integration and the formation of extracellular bone or tendon matrix. In this context, numerous studies suggest that osteoinductive substances such as bone morphogenetic protein-7 (BMP7) – which is already approved in the treatment of aseptic pseudoarthrosis – have a positive impact on bone-tendon healing. Yet, the cell biological and molecular biological processes during bone-tendon transition and specific effects of osteoinductive proteins remain unclear. The intention of this thesis was to establish a standardized co-culture model to simulate and investigate the osteoblast-fibroblast interaction. This new method was then applied to analyse the specific effect mechanisms of singular and repetitive stimulation with BMP7 on the bone-tendon healing. Based on the work of Wang et al. (2007), a standardized in vitro co-culture model was successfully established. It consists of secondary murine osteoblast and fibroblast cells incubated in a temporary two-chamber system. More precisely, an agarose divider served as a transient separator of osteoblastic and fibroblastic cells. After the removal of the divider direct interactions of both cell types were possible. Based on this model, the effects of singular and multiple stimulation of the osteoblastic and fibroblastic cells with recombinant human BMP7 (rhBMP7) were examined and analyzed to spot statistically significant changes. The study has shown that the established method provided an easy handling and at the same time showed that the co-culture model could be used as a high-throughput technique. Subsequently the histological analysis of the study showed a dose-dependent and number-dependent effect of the stimulation with rhBMP7. Hence, an accelerated migration and proliferation of the osteoblastic and fibroblastic cells after rhBMP7 application could be observed in the microscopical experiments. These observations were consistent with the results of the molecular biological examinations of specific osteogenic marker genes. Using qRT-PCR, you can see that stimulation with rhBMP7 leads to a dose-dependent increase of Alp1, Col1a1 and Bglap gene expressions in osteoblasts as well as an improved formation of bone matrix. An enhanced Spp1 gene expression as proposed in the common literature was not detectable for the osteoblastic cells in this study. The rhBMP7 application showed an increased gene expression for Runx2, Col1a1, Alp1 and Bglap in the examined fibroblastic cells. Surprisingly suggesting a transdifferentiation of fibroblasts into functional osteoblasts with developing osteoblast specific characteristics. The results of this study indicate that the bone-tendon integration on the one hand is achieved by enhanced bone matrix building of the osteoblasts and on the other hand by transdifferentiation of fibroblasts to functional osteoblastic cells. It seems that both cell types are responsible for the incorporation of tendon tissue into bone tissue including the formation of surrounding bone matrix. Since many cell biological and molecular biological processes of the bone-tendon integration remain unclear, further studies are required to generate a comprehensive understanding for the integration of tendons to bone and to ensure the development of reliable therapeutic approaches.