[Three-dimensional flow perfusion culture enhances proliferation of human fetal osteoblasts in large scaffold with controlled architecture]

Abstract

Objective: To demonstrate the feasibility and benefits of custom designed perfusion bioreactor in conjunction with well-defined three-dimensional (3D) environment for enhanced proliferation and homogeneous distribution of human fetal osteoblasts in large scaffold in vitro.

Methods: Large-scale β-tricalcium phosphate (β-TCP) scaffolds with tightly controlled architectures were fabricated. And a custom designed perfusion bioreactor was developed. Human fetal osteoblasts were seeded onto the scaffolds, cultured for up to 16 days in static or flow perfusion conditions. At Days 4, 8 & 16 post-incubation, the proliferation and distribution of osteoblasts were determined by daily D-glucose consumption, cell viability (methyl thiazolyl tetrazolium (MTT) assay), histological evaluation and scanning electron microcopy (SEM). Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray (EDX) analysis.

Results: In both static and perfusion cultures, the daily D-glucose consumption increased with prolonged time. The daily D-glucose consumption was significantly higher in the perfusion culture than that in static culture (P < 0.05). The increased cell viability with time during the culture was similar to the daily D-glucose consumption under both conditions. There was much greater cell viability under flow perfusion culture compared to static culture (P < 0.05). Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold. However, the cells were biased to periphery in scaffolds culture statically. Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis.

Conclusions: Flow perfusion culture plus well-defined 3D interconnected channel environments enhances the proliferation and improve the distribution of human fetal osteoblasts in large scaffolds. Scaffolds with controlled architecture may be a potential tool of studying the fluid flow configuration and cell behavior inside scaffold in details. And human fetal osteoblasts can be used as a cell source in large bone graft research.