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 freezedried, followed by cross-linking of combined dehydrothermal, glutaraldehyde, and ultraviolet treatment. Scanning electron microscopy revealed that collagen sponges exhibited homogeneous and interconnected pore structures with an average size of 180 microm, 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 osteogenic differentiation of MSCs in PGA-reinforced sponge at a weight ratio of 5 were greatly influenced by the culture method and growth conditions. Alkaline phosphatase (ALP) activity and osteocalcin 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.

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This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.

An ideal biomaterial for urethral reconstruction has not been developed. To create a urethral graft biomaterial with optimal biodegradability and biocompatibility, a copoly(L-lactide/epsilon-caprolactone) [P(LA/CL)] fabric tube was combined with a type I collagen sponge. The P(LA/CL) fibers were knitted into a vascular stent style (Type 1) or weaved into a mesh style (Type 2) to prepare P(LA/CL) tubes. The tubes were dipped in aqueous collagen solution and lyophilyzed to prepare the P(LA/CL)-collagen sponge graft. The grafts were applied to a 1.5-cm rabbit urethral defect (n = 14 for each condition), and tissue repair was evaluated using urethrographical, urethroscopical, and histological examination 1, 3, and 6 months after surgery. Although epithelialization was observed after 1 month in all Type 1 grafts, stenoses, fistulae, or stone formation was seen in 7 of the rabbits. In some cases, P(LA/CL) fibers prolapsed into the urethral lumen, causing stone formation. Only 3 rabbits survived for 6 months, and 2 of these had stenoses. For the Type 2 graft, all urethras were patent, without fistulae or stenoses, over the entire observation period. Histologically, urethral structure was disorganized for the Type 1 graft, whereas the urethral tissue on the Type 2 graft was slightly fibrotic but completely epithelialized and supported by a regenerated smooth muscle layer at 6 months. These findings suggest that creation of a scaffold suitable for urethral tissue regeneration will depend not only on the biomaterial composition, but also on the fabrication technique.