Flow perfusion co-culture of human mesenchymal stem cells and endothelial cells on biodegradable polymer scaffolds

Abstract

In this study, we investigated the effect of flow perfusion culture on the mineralization of co-cultures of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs). Osteogenically precultured hMSCs were seeded onto electrospun scaffolds in monoculture or a 1:1 ratio with HUVECs, cultured for 7 or 14 days in osteogenic medium under static or flow perfusion conditions, and the resulting constructs were analyzed for cellularity, alkaline phosphatase (ALP) activity and calcium content. In flow perfusion, constructs with monocultures of hMSCs demonstrated higher cellularity and calcium content, but lower ALP activity compared to corresponding static controls. ALP activity was enhanced in co-cultures under flow perfusion conditions, compared to hMSCs alone; however unlike the static controls, the calcium content of the co-cultures in flow perfusion was not different from the corresponding hMSC monocultures. The data suggest that co-cultures of hMSCs and HUVECs did not contribute to enhanced mineralization compared to hMSCs alone under the flow perfusion conditions investigated in this study. However, flow perfusion culture resulted in an enhanced spatial distribution of cells and matrix compared to static cultures, which were limited to a thin surface layer.

Figure 1. DNA content of constructs cultured for 14 days in osteogenic medium in static (St) or flow perfusion (Fl) conditions

Scaffolds were seeded with either 35,000 hMSCs (M1), 70,000 hMSCs (M2) or 35,000 hMSCs and 35,000 HUVECs (MH). Samples were taken after 0, 7 and 14 days of culture. Data are presented as means + standard deviation. At a given time point, statistical differences (p<0.05) between co-cultures (MH) and low-density monocultures (M1) are indicated by #, and differences (p<0.05) between co-cultures (MH) and high-density monocultures (M2) are indicated by *. At a given time point, statistical differences (p<0.05) between high-density monocultures (M2) and low-density monocultures (M1) are indicated by x, and statistical differences (p<0.05) between a flow perfusion group (Fl) and the corresponding static group (St) are indicated by +. Within a group, statistical differences (p<0.05) compared to previous time points are indicated by ‡.

Figure 2. ALP activity normalized to DNA in constructs cultured for 14 days in osteogenic medium in static (St) or flow perfusion (Fl) conditions

Scaffolds were seeded with either 35,000 hMSCs (M1), 70,000 hMSCs (M2) or 35,000 hMSCs and 35,000 HUVECs (MH). Samples were taken after 7 and 14 days of culture. Data are presented as means + standard deviation. At a given time point, statistical differences (p<0.05) between co-cultures (MH) and low-density monocultures (M1) are indicated by #, and differences (p<0.05) between co-cultures (MH) and high-density monocultures (M2) are indicated by *. At a given time point, statistical differences (p<0.05) between high-density monocultures (M2) and low-density monocultures (M1) are indicated by x, and statistical differences (p<0.05) between a flow perfusion group (Fl) and the corresponding static group (St) are indicated by +. Within a group, statistical differences (p<0.05) compared to previous time points are indicated by ‡.

Figure 3. Calcium content of constructs cultured for 14 days in osteogenic medium in static (St) or flow perfusion (Fl) conditions

Scaffolds were seeded with either 35,000 hMSCs (M1), 70,000 hMSCs (M2) or 35,000 hMSCs and 35,000 HUVECs (MH). Samples were taken after 7 and 14 days of culture. Data are presented as means + standard deviation. At a given time point, statistical differences (p<0.05) between co-cultures (MH) and low-density monocultures (M1) are indicated by #, and differences (p<0.05) between co-cultures (MH) and high-density monocultures (M2) are indicated by *. At a given time point, statistical differences (p<0.05) between high-density monocultures (M2) and low-density monocultures (M1) are indicated by x, and statistical differences (p<0.05) between a flow perfusion group (Fl) and the corresponding static group (St) are indicated by +. Within a group, statistical differences (p<0.05) compared to previous time points are indicated by ‡.

Figure 4. Representative histological sections of constructs cultured for 7 or 14 days in static (St) or flow perfusion (Fl) conditions

Scaffolds were seeded with either low (M1) or high (M2) densities of hMSCs, or co-cultures of hMSCs and HUVECs (MH). Samples were taken after 7 and 14 days of culture and were stained with Fast Green (green) to visualize the distribution of cells and matrix (first and third row) and von Kossa (brown) to visualize the presence of minerals (second and fourth row). Scale bar represents 100 µm in all images.

Figure 5. Scanning electron micrographs of the top surfaces of constructs cultured for 7 or 14 days in static (St) or flow perfusion (Fl) conditions

Scaffolds were seeded with either low (M1) or high (M2) densities of hMSCs, or co-cultures of hMSCs and HUVECs (MH). Samples were taken after 7 and 14 days of culture. Panels A and C are magnified at 300x, and the scale bar is 200 µm and applies for all images in these panels. Panels B and D are magnified at 1600x, the scale bar is 30 µm and applies for all images in these panels.