Kurzfassung

Clinical studies revealed that large amounts of tissue engineered bone material fail to heal, obviously due to insuf-ficient oxygen and nutrient supply. Blood vessel invasion into the engineered bone construct seems to be the most important requirement for its survival. But, angiogenesis and osteogenesis depend on a closed interaction between endothelial cells and osteoblasts. However, less attention has been paid to endothelial cells as potential candidates for bone tissue engineering so...Clinical studies revealed that large amounts of tissue engineered bone material fail to heal, obviously due to insuf-ficient oxygen and nutrient supply. Blood vessel invasion into the engineered bone construct seems to be the most important requirement for its survival. But, angiogenesis and osteogenesis depend on a closed interaction between endothelial cells and osteoblasts. However, less attention has been paid to endothelial cells as potential candidates for bone tissue engineering so far. The aim of the study is to evaluate cell interaction between endothelial progeni-tor cells from peripheral blood and primary osteoblasts cultured on biomaterials. The project will help to clarify the hypothesis that co-cultures of hOB/EPC are useful to improve the quality of tissue engineered bone constructs.

In the first part of experiments, endothelial differentiation of outgrowth cultures of EPC from buffy coats of venous blood samples is induced under endothelial culture conditions. Cell numbers and phenotypes are characterized by FACS using antibodies against specific cell markers. Purified cultures of EPC (CD31+/vWF+) are isolated by MACS for further experiments. In the second part of the project, cell function and differentiation of EPC/pOB co-cultures is compared to the monotypic control cultures. Three dimensional cultures are introduced by using bioma-terials from Biomet. Cell proliferation and viability are assayed using the photometric MTT-test and fluorimetric DNA-content measurement with Hoechst-33248. Angiogenic differentiation of EPC is analyzed by immunohistol-ogy and FACS for CD31, CD34, and vWF. Osteogenic differentiation of pOB is assayed by measurement of alka-line phosphatase activity, and qPCR for collagen type 1, Cbfa1, and osteocalcin. Sprouts and neo-vessel formations are detected by quantitative measurement of vWF-positive tubuli using Leica-image analysis system. Future ex-periments will be necessary to evaluate the in-vivo development of EPC/pOB after implantation in nude mice
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