In vitro dynamic perfusion of prevascularized OECs-DBMs (outgrowth endothelial progenitor cell - demineralized bone matrix) complex fused to recipient vessels in an internal inosculation manner

Abstract

The current research on seed cells and scaffold materials of bone tissue engineering has achieved milestones. Nevertheless, necrosis of seed cells in center of bone scaffold is a bottleneck in tissue engineering. Therefore, this study aimed to investigate the in vivo inosculation mechanism of recipient microvasculature and prevascularized outgrowth endothelial progenitor cells (OECs)-demineralized bone matrix (DBM) complex. A dorsal skinfold window-chamber model with tail vein injection of Texas red-dextran was established to confirm the optimal observation time of microvessels. OECs-DBM complex under static and dynamic perfusion culture was implanted into the model to analyze vascularization. OECs-DBM complex was harvested on 12th day for HE staining and fluorescent imaging. The model was successfully constructed, and the most appropriate time to observe microvessels was 15 min after injection. The ingrowth of recipient microvessels arcoss the border of OECs-DBM complex increased with time in both groups, and more microvessels across the border were observed in dynamic perfusion group on 3rd, 5th, 7th day. Fluorescent integrated density of border in dynamic perfusion group was higher at all-time points, and the difference was more significant in central area. Fluorescent imaging of OECs-DBM complex exhibited that no enhanced green fluorescent protein-positive cells were found beyond the verge of DBM scaffold in both groups. In vitro prevascularization by dynamic perfusion culture can increase and accelerate the blood perfusion of OECs-DBM complex obtained from recipient microvasculature by internal inosculation. Accordingly, this approach may markedly contribute to the future success of tissue engineering applications in clinical practice.

Keywords: Dynamic perfusion culture; decalcified bone matrix; internal inosculation; outgrowth endothelial progenitor cells; vascularization.

Graphical abstract

Figure 1.

Dorsal skinfold window-chamber assembling on BABL/C nude mice.

Figure 2.

Implanting of OECs-DBM complex and illustration of implanting layers.

Figure 3.

Observation platform for nude mice burdening dorsal skinfold window-chamber.

Figure 4.

Determination of time window for observation of vascularization. Scale bar: 1 mm.

Figure 5.

Observation and fluorescent imaging of microvessels. a: Microvessels under light microscopy prior to injection of Texas red-dextran; b: Microvessels under light microscopy after injection of Texas red-dextran; c: Microvessels under fluorescence microscope after injection of Texas red-dextran. Scale bar: 100 μm.

Figure 6.

Dynamic observation of vascularization in OECS-DBM complex. a: Ingrowth of recipient microvessels across the verge of OECs-DBM complex. Scale bar: 100 μm. Dot line represents verge of OECs-DBM complex, white arrow represents recipient microvessel grows into OECs-DBM complex. b: Number of ingrown microvessels–time curve. Compare with OECs-DBM scaffold under dynamic culture, *P < 0.05.

Figure 7.

OECS-DBM complex was observed in static culture and dynamic culture group. a: In vivo observation and fluorescent imaging of OECs-DBM complex under static culture (left) and dynamic culture (right). Scale bar: 1 mm. b: Integrated density of scaffold border–time curve. c: Integrated density of scaffold central area–time curve. Compare with OECs-DBM scaffold under dynamic culture, *P < 0.05, **P < 0.01.

Figure 8.

HE staining and fluorescent imaging of OECs-DBM scaffold slice under static culture and dynamic culture. Dot line represents verge of scaffold, red arrow represents OECs lumps, white arrow represents inflammatory cells, black arrow represents microvessels from recipient. Scale bar: 200 μm.