Abstract
There is a great need for strategies to fill large bone defects, to treat non-healing bone fractures and to merge vertebrae. Local application of growth factors, such as BMP-2 (bone morphogenetic protein) is one of the promising strategies. BMP-2 plays an important role in inducing osteogenic differentiation of stem cells.
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It can induce large amounts of bone formation when applied locally in patients. However, in the clinical situation extremely high dosages of BMP are needed due to quick wash out and degradation by proteinases. These high dosages are likely the cause of side effects, that are frequently observed and which can be life threatening. As a result, application of BMP-2 is only justified in certain severe cases. In this thesis we investigate new methods in which prolonged BMP-2 presence can be accomplished. The goal is to induce a long-term delivery of low doses the specific protein, thereby mimicking it’s natural presence as observed in fracture healing. Our research focuses on inducing bone formation by non-viral gene delivery, using the BMP-2 gene. We use non-viral strategies, as they are considered safe compared to viral strategies. First, we investigate the application of local non-viral gene delivery without the addition of cells. DNA coding for the BMP-2 gene is applied with the aim to stimulate the endogenous cells, which are present at the location of implantation, to produce BMP-2. We show that the cells present in situ are indeed able to take up the DNA and produce BMP. However, bone formation is not achieved. Second, we demonstrate that DNA-BMP-2 can be combined with DNA coding for other proteins from the BMP family: DNA-BMP-6 or -7. These combinations of DNA appear to lead to the formation of heterodimers with a much stronger osteogenic effect than BMP-2 homodimers. Third, we investigate gene delivery with cells. In this case, harvested cells from the patient that are treated with BMP genes and subsequently implanted at the desired location. As some cell types have large influence on the bone formation process, we compare bone stem cells with non-osteogenic cells. Against the expectation, we show in animal experiments that comparable amounts of bone formation are formed. From this we conclude that not only stem cells but also other cell types are suitable as BMP producers and that the actual bone formation takes mainly place by the patient’s own cells. Lastly, our research focuses on the application of other, easier to harvest cells, such as adipose tissue cells or bone marrow cells. We show that it is possible to isolate these cells, treat them with the desired genes and implant them within the time frame of a few hours. The desired result, bone formation, is achieved. This makes the step toward the application of gene delivery within a single procedure small.
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