Kurzfassung

Clinical applications of bone tissue engineering have been limited due to a shortage of critical oxygen and nutrient supply post-implantation. Improving vascularization by implanting endothelial cells may represent a key solution for engineering bone constructs of large size. Based on the promising preliminary data concerning the role of CD34+- endothelial progenitor cells (EPC) in fracture healing, we investigated cell interactions and differentiation of CD34+-EPC isolated from peripheral...Clinical applications of bone tissue engineering have been limited due to a shortage of critical oxygen and nutrient supply post-implantation. Improving vascularization by implanting endothelial cells may represent a key solution for engineering bone constructs of large size. Based on the promising preliminary data concerning the role of CD34+- endothelial progenitor cells (EPC) in fracture healing, we investigated cell interactions and differentiation of CD34+-EPC isolated from peripheral blood of severely injured patients and human adult osteoblasts (hOB) in a three dimensional environment using mineralized collagen scaffolds relevant for bone tissue engineering. In this project we focused on the effect of cell-cell-contact on cell proliferation and expression of endothelial and osteoblastic marker genes.
In a population of patients with multiple injuries who are potential candidates for tissue engineering approaches (ISS-Score > 16 pts.) we could clearly show that the number of CD34+-EPC increases significantly at days 1, 3, and 7 after the polytrauma as compared to a sex- and age matched control population. Cells expressing the surface antigen PECAM-1 (CD31, mature endothelial cells) were also significantly elevated in peripheral blood of those patients; whereas the marker of bone marrow endothelial cells CD133+ remained almost not detectable. Results of a well established angiogenesis assay revealed that long term monocultures of CD34+-EPC undergo apoptosis and are not able to survive without additional stimulation with angiogenic factors. Using these model we could clearly show that both cell proliferation and endothelial sprout formation of CD34+-cells were significantly induced by hOB. Co-cultures of CD34+ cells and hOB were characterized by vital cell growth within the scaffolds. Lumen-formation as a typical feature of endothelial cell differentiation, however, was only detected in control HUVEC/LV-eGFP-hOBs co-cultures.
In order to analyze cell-specific regulation of gene expression we established co-cultures using osteoblasts transfected with the SEW-eGFP-lentiviral vector. This vector not only allows the visualization of the cells by eGFP fluorescence, but also enables quantification of gene expression in each cell type by correction for eGFP-mRNA, which is selectively and stably expressed in osteoblasts. Using this accurate model we could verify different effects of primitive CD34+-progenitor and more mature HUVE-cells on human osteoblasts. With exception of the transcription factor Runx2, HUVECs were able to induce expression of different osteoblast marker genes (e.g. alkaline phosphatase, osteopontin, SP7). In clear contrast, CD34+-EPC did not significantly affect expression of osteoblast markers in co-cultures. On the other hand, hOB significantly induced expression of endothelial marker genes PECAM-1, CD146, and vWF both in CD34+-EPC and HUVECs.

This study provides new insight into the cellular interactions between CD34+-EPC and hOB and helps assess the significance of these cells for potential applications in bone tissue engineering and fracture healing. Using our model, accurate analysis of gene expression in different cell types will be possible for different tissue engineering applications in the future.
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