Flow perfusion culture of human fetal bone cells in large beta-tricalcium phosphate scaffold with controlled architecture.

One unsolved problem in bone tissue engineering is how to enable the survival and proliferation of osteoblastic cells in large scaffolds. In this work, large beta-tricalcium phosphate scaffolds with tightly controlled channel architectures were fabricated and a custom-designed perfusion bioreactor was developed. Human fetal bone cells in third passage were seeded onto the scaffolds and cultured in static or flow perfusion conditions for up to 16 days. Compared with nonperfused constructs, flow perfused constructs demonstrated improved cells proliferation and differentiation according to cell viability, glucose consumption, alkaline phosphatase activity, and osteopontin. Moreover, after 16 days of perfusion culture, a homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observed by scanning electron microscopy and histological study. In contrast, cells were located only along the scaffold perimeter in static culture. These results demonstrated the feasibility and benefit of perfusion culture in conjunction with well-defined three-dimensional environment for large bone graft construction. Porous scaffold with controlled architecture can be a potential tool to evaluate the effects of scaffold specific geometry on fluid flow configuration and cell behavior under perfusion culture.

[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].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Fabrication of scaffold with controlled porous structure and flow perfusion culture in vitro].

3D Scaffolds with controlled porous structure were designed and fabricated by utilizing CAD and rapid prototyping techniques. A flow perfusion bioreactor, which allowed exposure of the culture cells to low levels of mechanical stimulation by fluid flow-induced shear stress, was developed in our lab. The scaffolds were pre-designed and the negative images of the designs were used to build the molds on a stereolithography (SL) apparatus with epoxy resins. Calcium phosphate cement paste was cast into the molds. And after pyrolysis, the 3D scaffolds with controlled internal pore architectures were obtained. Rabbit osteoblasts were seeded in 3D porous scaffolds, cultured in the flow perfusion bioreactor with media flow rate set at 2 mL/min and 6-well plates. At 3, 7, and 14 days, scanning microscopic evaluation showed excellent growth on the surface of scaffolds and poor viability of cells within microchannels in static cultures. In flow perfusion bioreactor, there was greater cellularity throughout the scaffolds and abundant deposition of extracellular matrix. Cells were also seen throughout the internal microchannels of scaffolds. These results represent that better mass transport of oxygen and nutrient occurred in the flow perfusion bioreactor and cells distribution in 3D porous scaffolds was improved.