1476 INTRODUCTION F OR SUCCESSFUL TISSUE REGENERATION, cells consti- tuting the tissue to be regenerated, such as mature, progenitor, and precursor cells, are necessary. Consid- ering the proliferation activity and differentiation po- tential of cells, stem cells are the most promising, in a practical sense. Among them, mesenchymal stem cells (MSCs) have been widely investigated for use by them- selves or combined with scaffolds necessary for pro- motion of cell proliferation and differentiation. It was found that MSCs are inherently able to differentiate into not only osteogenic lineage cells but also into chon- drogenic, myogenic, adipogenic, and neurogenic lin- eages. 1–5 MSCs have been experimentally used to dem- onstrate their in vivo potential to induce the regeneration of mesenchymal tissues. 6–10 Since the publication of re- ports that these cells are effective in inducing the re- generation of tissues other than mesenchyme, 11–13 their feasibility as a cell source for regenerative medicine is anticipated. They are, in addition, isolated from patients themselves. TISSUE ENGINEERING Volume 11, Number 9/10, 2005 © Mary Ann Liebert, Inc. Perfusion Culture Enhances Osteogenic Differentiation of Rat Mesenchymal Stem Cells in Collagen Sponge Reinforced with Poly(Glycolic Acid) Fiber HOSSEIN HOSSEINKHANI, Ph.D., YASUYUKI INATSUGU, B.Eng., YOSUKE HIRAOKA, M.Agr., SACHIKO INOUE, M.Eng., and YASUHIKO TABATA, Ph.D., D.Med.Sci., D.Pharm. ABSTRACT The objective of this study was to obtain fundamental knowledge about in vitro culture systems to enhance the proliferation and differentiation of mesenchymal stem cells (MSCs) in collagen sponge reinforced by the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously localized at PGA:collagen weight ratios of 0.67, 1.25, 2.5, and 5 was freeze- dried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treat- ment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and in- terconnected pore structures with an average size of 180 m, irrespective of PGA fiber incorporation. When rat MSCs were seeded into collagen sponge with or without PGA fiber incorporation, more attached cells were observed in collagen sponge incorporating PGA fiber than in collagen sponge without PGA fiber incorporation, irrespective of the PGA:collagen ratio. The proliferation and os- teogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly in- fluenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and os- teocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method became maximum at a flow rate of 0.2 mL/min, although they increased with culture time period. It may be concluded that appropriate perfusion conditions enable MSCs to positively improve the extent of proliferation and differentiation. Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.