Analysis of the balance between proliferation and apoptosis of cultured vascular smooth muscle cells for tissue-engineering applications

Tissue homeostasis, the balance between cell proliferation and apoptosis, is an important factor in tissue engineering. We describe a new method to analyze markers of both proliferation and apoptosis in a single assay to monitor growth behavior of cell cultures. Human vascular smooth muscle cells (VSMCs) were cultured either on gelatin-coated tissue culture polystyrene or in three-dimensional porous scaffolds composed of insoluble collagen and elastin. mRNA concentrations of cyclin E, as a marker of proliferation, and of tissue transglutaminase (tTG) as a marker of apoptosis, quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to porphobilinogen deaminase mRNA concentrations, were analyzed. tTG mRNA expression levels were increased when apoptosis was induced by tumor necrosis factor-α in combination with cycloheximide or by culturing the cells in serum-free culture medium. Cyclin E mRNA expression levels were less altered in these cell cultures. Results were compared with several reference tests to measure apoptosis including DNA fragmentation, annexin V staining, and light microscopy. This RT-PCR method could be used to characterize cell growth behavior of VSMCs in vitro. In addition, it was shown that this test is suitable to measure the balance between proliferation and apoptosis of VSMCs present in tissue-engineered constructs.