Samenvatting

Bone tissue engineering is an interesting research field for the treatment of large bone defects and bone related diseases. Mesenchymal stromal cells are an attractive cell source to use in bone tissue engineering. In animal models the combination of mesenchymal stromal cells and scaffolds showed increased bone formation and fracture healing. Bioluminescent imaging is an attractive tool to monitor in vivo bone formation, because it makes it possible to monitor real time reactions without the need to sacrifice animals. The technique is based on the activity of the enzyme luciferase, which catalyses the reaction of the substrate D-luciferin into oxyluciferin, which leads to the emission of photons. This light can be detected with a very light sensitive camera, called bioluminescent imaging. Mesenchymal stromal cells are multipotent cells, they can differentiate in vitro into osteoblasts, chondrocytes, adipocytes, myocytes and tendon fibroblasts. They are isolated from different sources, but mainly from the bone marrow. They can be easily isolated because of their plastic adherence, and expanded in culture in the presence of growth factors and cytokines. A luciferase gene driven by a constitutive promoter can be used to monitor the proliferation of transduced mesenchymal stromal cells. A luciferase gene driven by a bone tissue specific promoter (osteocalcin or collagen type I) could be used to monitor the differentiation of the mesenchymal stromal cells during bone formation. Lentiviral transduction, has been used for a stable integration of the promoter with the transgene in the mesenchymal stromal cells. Titrations showed that the optimal conditions for transduction is a 24 hour incubation and a polybrene concentration of 6 μg/ml. The transduction efficiency was determined, according to the percentage of green fluorescence protein positive cells, using fluorescence activated cell sorting. The green fluorescence protein expression is stable integrated in the genome and showed longterm expression in time. Transduced mesenchymal stromal cells are still able to differentiate into adipocytes and osteoblasts. However, the luciferase activity decreases after osteoblast differentiation. This could be due to a lower metabolism of the cells. To test the functionality of the LV-OC-Luc2-CMV-GFP construct; human mesenchymal stromal cells were transduced and induced with vitamin D3. The stimulation with vitamin D3 resulted in a 1.7 ± 0.3 fold induction of luciferase expression. Previous experiments showed that it is not possible to transduce human mesenchymal stromal cells in the presence of platelet lysate or human serum albumin. Pre-conditioning of the cells with one of the following methods, an acidic wash on a cell pellet or on the cells in the flask, trypsinisation of the cells or 10 times washing with phosphate buffered saline before the transduction makes it possible to transduce cells which are cultured in platelet lysate medium. Bioluminescent imaging has, after optimalisation, the potential to monitor and quantify the dynamic process of bone production and to study underlying molecular mechanisms