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Cardiomyocytes in the 2D were observed to attach more closely than in the 3D nanopeptide scaffolds (Figure?2, A). The cells were round shape in 2D and elongated, spindle shape in 3D culture ( Figure?2, B, C). However, because of the cells were embedded in gel-like 3D scaffolds and this website spread stereoscopically, the global alignment and cell-cell interaction of cardiomyocytes in 3D culture are difficult to observe by fluorescence microscope. Thus we used scanning electron microscopy (SEM) to exhibit the cardiomyocyte allocation in 3D nanopeptide scaffolds. Similar to the SEM observation of PuraMatrix in other group, 15 the expected nanofibers of 0.5% PuraMatrix range from 5 to 30?nm, approximate extracellular matrix environment. The cardiomyocytes were truly embedded in the nanopeptide scaffold, spread and contacted each other less orderly ( Figure?3). These images proved the nanopeptide scaffold embedded cells in 3D microenvironment. Although Fluvoxamine the cardiomyocytes remained spontaneous beating in both groups, cardiomyocytes in the 3D group seemed to show an irregular beating frequency when observed by direct microscopy. For further examination, dual excitation fluorescence measurement system (IonOptix) was used to measure intracellular calcium transients of a single cardiomyocyte on culture day 3. The cardiomyocytes in the 2D system retained a consistent beating pattern (Figure?4, A); while the cardiomyocytes in the 3D system had irregular beating rhythm ( Figure?4, B). Further analysis demonstrated that cardiomyocytes in the 3D system had an approximate 1.42-fold increase in basal level of intracellular calcium concentration as compared with the cardiomyocytes from the 2D culture group ( Table?1). The amplitude of calcium transient was smaller as well as the duration of calcium transient was longer in 3D cells as compared with those in 2D cells. Additionally, the culture medium of cardiomyocytes from the 3D culture group contained higher BNP levels than did that of cardiomyocytes from the 2D culture group (197.60?��?57.86 vs. 59.59?��?12.44?pg/ml, p?Pazopanib concentration expression levels corresponding to the T-type calcium channels Cav3.1 and Cav3.2, the L-type calcium channel CANAC1, and the gap junction protein CX 43 were evaluated by mRNA RT-PCR after 3?days in culture (Table?2). Results are expressed as the fold change relative to the expression level of the cardiomyocytes in the standard 2D culture. No significant differences were observed in T-type or L-type calcium channel expression levels between cardiomyocytes from the 3D culture and the 2D control group. CX 43 expression was increased 2.62-fold in the cardiomyocytes from the 3D culture relative to the cardiomyocytes from the 2D culture.