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Journal of Biochemistry Advance Access originally published online on March 23, 2007
Journal of Biochemistry 2007 141(5):729-736; doi:10.1093/jb/mvm077
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© 2007 The Japanese Biochemical Society.

In vitro Mineralization by Mesenchymal Stem Cells Cultured on Titanium Scaffolds

Masahiko Maeda1,2, Motohiro Hirose2,*, Hajime Ohgushi2 and Tadaaki Kirita1

1Department of Oral and Maxillofacial Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521; and 2Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan

*To whom correspondence should be addressed. Tel: +81-6-6494-7807, Fax: +81-6-6494-7861, E-mail: motohiro-hirose{at}aist.go.jp

Received December 3, 2006; Accepted February 27, 2007


  Abstract

Titanium has been utilized in the field of orthopaedic and dental reconstructive surgery, but mineralization through osteogenic differentiation of osteogenic cells on titanium surfaces has not been fully investigated. Here we cultured rat mesenchymal stem cells (MSCs) on the surfaces of titanium dishes in osteogenic media containing calcein which is a calcium-binding fluorescence dye. On titanium dishes, MSCs showed high viability to adhere to the surfaces and excellent proliferation. At day 14 of culture, MSCs differentiated into osteoblasts to form mineralized matrices on titanium dishes as well as tissue culture polystyrene (TCPS) dishes which are widely recognized as optimal culture substrates. Calcein was incorporated into the bone minerals fabricated by MSCs cultured on both substrates to show green emission under fluorescence microscopy. The fluorescence intensity was quantified with an image analyser during culture periods. These results indicate that the surfaces of titanium showed a high adhesion/proliferation potential to MSCs and that the titanium effectively supported the osteogenic differentiation of MSCs comparable to TCPS dishes. Therefore, the titanium is an effective scaffold that is applicable in bone reconstruction surgery.

Key Words: calcein, cell adhesion, mesenchymal stem cell, osteogenic differentiation, titanium

Abbreviations: calcein-AM, calcein-bis [(acetyloxy) methyl] ester; Calcein F. I., calcein fluorescence intensity; Dex, dexamethasone; EDX, energy dispersive X-ray spectroscopy; EthD-1, ethidium homodimer-1; FBS, fetal bovine serum; hFbs, human dermal fibroblasts; MEM, minimum essential medium; MSCs, mesenchymal stem cells; PBS(–), calcium/magnesium-free phosphate-buffered saline; P-1, passage-1; P-3, passage-3; Ra, surface roughness; SEM, scanning electron microscopy; TCPS, tissue culture polystyrene


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