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Biotechnol. Appl. Biochem. (2006) 45, (65–74) (Printed in Great Britain)

Fabrication and detection of tissue-engineered bones with bio-derived scaffolds in a rotating bioreactor

Kedong Song*, Zhiming Yang†, Tianqing Liu*1, Wei Zhi†, Xiangqin Li*, Li Deng†, Zhanfeng Cui‡ and Xuehu Ma*

*Stem Cell and Tissue Engineering Laboratory, Dalian University of Technology, Dalian 116024, People's Republic of China, †Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotecherapy, West China Hospital, Sichuan University, Sichuan 610041, People's Republic of China, and ‡Department of Engineering Science, University of Oxford, Parks Road, Oxford OX1 3PJ, U.K.

Key words: bio-derived scaffold, green fluorescent protein, osteoblast, rotating bioreactor, three-dimensional tissue culture, tissue-engineered bone.

Abbreviations used: ALP, alkaline phosphatase; BDBS, bio-derived bone scaffolds; BMP-2, bone morphogenetic protein-2; DAPI, 4´,6-diamidino-2-phenylindole; FBS, fetal bovine serum; GFP, green fluorescent protein; H/E, haematoxylin/eosin; HMDS, hexamethyldisilazane; PLGA, poly(lactic acid-co-glycollic acid); RWVB, rotating-wall vessel bioreactor; SEM, scanning electron microscope.

¹To whom correspondence should be addressed (email liutq@dlut.edu.cn).

In order to explore the methods for commercialized bone tissue engineering, engineered bones should be cultivated in bioreactors to realize three-dimensional culture under well-defined culture conditions. In the present paper, osteoblasts isolated from the cranium of 1-month-old Zelanian rabbits were inoculated on to the BDBS (bio-derived bone scaffolds) to investigate the three-dimensional fabrication of engineered bone in an RWVB (rotating-wall vessel bioreactor). The osteoblasts, after being transfected with green fluorescent protein, were respectively seeded at 2×10⁶ and 1×10⁶ cells·ml^-1 on to the BDBS and then cultured in a T-flask and an RWVB for 1 week. The morphologies and structure of the fabricated bone were investigated by using an inverted microscope, a scanning electron microscope and a laser confocal microscope using the stains haematoxylin/eosin and Toluidine Blue. After being digested from the scaffolds, the cells were assayed with ALP (alkaline phosphatase) stain, von-Kossa staining on mineralized nodules, type I collagen and bone morphogenetic protein-2 expression, and the cell expansion and growth curves using different culture methods were quantitatively determined with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Furthermore, cell cycle and apoptosis were detected by using a flow cytometer, and total DNA was also assayed. For a comparative study, cell-seeded constructs were also cultured under static conditions. The results show that the cell number cultured in the RWVB was five times that in the T-flask. Bone tissues cultured in the RWVB with two different densities grew well, and the osteoblasts maintained their normal cycle and DNA content. The result demonstrates that, with the stress stimulation in the fluid in the RWVB, the active expression of ALP can be increased, rapid proliferation and differentiation of osteoblasts are possible and the three-dimensional fabrication of engineered bone could be realized.

Received 3 March 2006/4 May 2006; accepted 9 May 2006

Published as Immediate Publication 9 May 2006, doi:10.1042/BA20060045

© 2006 Portland Press Ltd