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.

This article describes an investigation of collagen sponge mechanically reinforced through the incorporation of poly(glycolic acid) (PGA) fiber. A collagen solution with PGA fiber homogeneously dispersed at collagen:PGA weight ratios of 1.5, 0.8, 0.4, and 0.2 was freeze-dried, followed by dehydrothermal cross-linking to obtain collagen sponges incorporating PGA fiber to various extents. By scanning electron microscopy observation, the collagen sponges exhibited isotropic and interconnected pore structures with an average size of 180 microm, irrespective of PGA fiber incorporation. As expected, PGA fiber incorporation enabled the collagen sponges to significantly enhance their compression strength. In vitro cell culture studies revealed that the number of L929 fibroblasts initially attached was significantly greater for any collagen sponge incorporating PGA fiber than for collagen sponge. The shrinkage of sponge after cell seeding was suppressed by fiber incorporation. It is possible that shrinkage suppression results in the superior cell attachment of sponge incorporating PGA fiber. After subcutaneous implantation into the backs of mice, the residual volume of collagen sponge incorporating PGA fiber was significant compared with that of collagen sponge and increased with a decrease in the collagen:PGA ratio. The greater number of cells infiltrated and deeper infiltration were observed for collagen sponge incorporating PGA fiber implanted subcutaneously. We conclude that the incorporation of PGA fiber is a simple and promising way to reinforce collagen sponge without impairing biocompatibility.

Engineering the cellular microenvironment has great potential to create a platform technology toward engineering of tissue and organs. This study aims to engineer a neural microenvironment through fabrication of threedimensional (3D) engineered collagen matrixes mimicking in-vivo-like conditions. Collagen was chemically modified with a pentapeptide epitope consisting of isoleucine-lysine-valine-alanine-valine (IKVAV) to mimic laminin structure supports of the neural extracellular matrix (ECM). Three-dimensional collagen matrixes with and without IKVAV peptide modification were fabricated by freeze-drying technology and chemical cross-linking with glutaraldehyde. Structural information of 3D collagen matrixes indicated interconnected pores structure with an average pore size of 180 μm. Our results indicated that culture of dorsal root ganglion (DRG) cells in 3D collagen matrix was greatly influenced by 3D culture method and significantly enhanced with engineered collagen matrix conjugated with IKVAV peptide. It may be concluded that an appropriate 3D culture of neurons enables DRG to positively improve the cellular fate toward further acceleration in tissue regeneration. KEYWORDS: Tissue engineering, 3D matrix, peptide, collagen, IKVAV E ngineering the cellular microenvironment is very important to fabricate three-dimensional (3D) models toward better understanding of cell−tissue interactions and regenerative medicine technology.1−5 Biodegradable materials are at the core of fabrication of 3D engineered tissues together with cell culture technology. Three-dimensional in vitro technology aims to create a platform filed that are suitable for cell−cell interactions as they do in vivo. We are not able to mimic these interactions in common tissue culture dishes or 2D in vitro culture systems. Therefore, such an engineered design would be necessary to address the above challenges. Natural extracellular matrix (ECM) plays important roles in creating microenvironment for cell−cell as well as cell−tissue interactions. Therefore, 3D in vitro models should have similar structure as ECM does. These similarities are in terms of biological, chemical, and physical composition. Several 3D structures have been already developed for cell culture in scaffolds since they provide larger surface area for cell attachment and proliferation than 2D tissue culture dishes. 6−10This study aims is to establish a simple 3D in vitro platform technology to analyze the proliferation capability of dorsal root ganglion (DRG) cells under in-vivo-like conditions. We characterized and studied cellular behavior of DRG cells by testing their potential proliferation by culturing them in an invivo-like condition. The data described in the current study suggests that this approach may be widely applicable to many stem cell populations. In the present technology, the molecular design of such an in-vivo-like condition was undertaken to design a 3D collagen matrix incorporated the pentapeptide epitope isoleucine-lysine-valine-alanine-valine (IKVAV)...

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