Rotating three-dimensional dynamic culture of osteoblasts seeded on segmental scaffolds with controlled internal channel architectures for construction of segmental tissue engineered bone in vitro

Abstract

To explore the feasibility, effectiveness, and mechanism of culturing osteoblasts on calcium phosphate cement (CPC) scaffolds with controlled internal channel architectures in a rotating bioreactor, and to develop a novel method for construction of segmental tissue engineered bone in vitro. Self-hardening CPC scaffolds with controlled internal channel architectures were designed and fabricated using computer aided design (CAD) and indirect rapid prototyping (RP) techniques. A rotating bioreactor was developed. Osteoblasts were isolated from the skull of rabbit and seeded onto the CPC scaffolds, cultured for up to 21 days in static or rotating three-dimensional (3D) dynamic conditions. 7, 14, and 21 days after the incubation the proliferation, metabolic activity, and differentiation of the osteoblasts were determined by MTT, glucose consumption, and alkaline phosphate activity (ALP) assays respectively. The distribution of cells throughout the scaffolds was observed by scanning electron microscopy (SEM) and the sphere like structures which the SEM images showed within the extracellular matrix were assessed by energy dispersive X-ray (EDX) analysis. At all culture time points, the rotatingly cultured constructs demonstrated greater proliferation, metabolic activity, and osteoblastic differentiation than those of the statically cultured constructs as evidenced by MTT, glucose consumption and ALP assays. SEM indicated that 21 days after the distribution of cells in the scaffolds in the rotating culture was much more uniform than in the static culture. The sphere like structures was identified as calcium phosphate nodules by EDX analysis. As a novel method for construction of segmental tissue engineered bone in vitro, the rotating 3D dynamic culture of osteoblasts on CPC scaffolds with controlled internal channel architectures improves the properties such as proliferation, metabolic activity, osteoblastic differentiation, and uniform distribution of the seeded cells over those that maintain in static culture.