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Biomacromolecules 2010, 11, 1948–1955
Investigation of Pore Size Effect on Chondrogenic Differentiation of Adipose Stem Cells Using a Pore Size Gradient Scaffold Se Heang Oh,† Tae Ho Kim,† Gun Il Im,‡ and Jin Ho Lee*,† Department of Advanced Materials, Hannam University, 461-6 Jeonmin Dong, Yuseong Gu, Daejeon 305-811, Republic of Korea, and Department of Orthopedics, Dongguk University International Hospital, 814 Sigsa Dong, Goyang 411-373, Republic of Korea Received February 22, 2010; Revised Manuscript Received June 23, 2010
In this study, polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by the centrifugation and thermal fibril-bonding process. The fabricated PCL scaffold showed a gradual increasing pore size (from ∼90 to ∼400 µm) and porosity (from ∼80 to ∼97%) along the cylindrical axis. The pore size gradient PCL scaffold was used to investigate the effect of pore size on the chondrogenic differentiation of adipose stem cells (ASCs). From the in vitro culture of ASCs, it was observed that the scaffold section having a pore size range of 370-400 µm provided a more favorable environment for chondrogenic differentiation than other pore size groups. The pore size gradient scaffolds can be a good tool for the systematic study of determining optimum pore size ranges for a variety of stem cell differentiation to a specific cell type.
Introduction Tissue engineering with the use of stem cells that can be selfregenerated and can be expanded to high cell numbers as well as differentiated into certain cell types is a rapidly emerging field for the regeneration or reconstruction of a variety of tissues and organs.1-5 It is well recognized that the efficient stem cell differentiation into a target cell is an essential requirement for clinical applications. To effectively induce the differentiation of the stem cells, the chemical cues including growth factors have been commonly adapted, and encouraging outcomes have been reported.6 Although it is widely believed that the physical cues, such as pore size and porosity in three-dimensional (3-D) scaffolds, can also influence the cell differentiation,6 there is scattered data available indicating which pore size or porosity is favorable for stem cell differentiation into a specific cell type. Many research groups have reported the different optimal pore sizes or porosity ranges for bone marrow stem cell (BMSC) differentiation to target cells/tissues. For example, the pore size (porosity) ranges of 200 µm (74%),7 200-500 µm,8 and 860 µm (60%)9 were reported to be desirable for chondrogenic differentiation; those of 40 µm (30%),10 150-200 µm (36%),11 200 µm (75%),12,13 250 µm (86%),14 and 450 µm (70%)15 were reported to be suitable for osteogenic differentiation; and the pore size range of 50-200 µm16 was also reported to allow appropriate smooth muscle differentiation. Recently, a few research groups have also reported optimal pore size (porosity) ranges for adipose stem cell (ASC) differentiation to target cells/ tissues; the pore size (porosity) ranges of 100-125 µm (89-92%)17 and 200-300 µm18 were reported to provide a suitable environment for chondrogenic differentiation, while the pore size range of 120-200 µm19 proved to be desirable for hepatic differentiation. These diverse optimal pore size (porosity) * To whom correspondence should be addressed. Tel.: +82-42-629-8859. Fax: +82-42-629-8854. E-mail:
[email protected]. † Hannam University. ‡ Dongguk University.
ranges suggested for the differentiation to a specific cell or tissue may be caused by the different scaffold fabrication conditions, which are the result of the different pore architectures in the scaffolds, as well as different cell cultures and differentiation conditions. Therefore, a series of scaffolds with wide ranges of pore sizes are still required to determine the optimum pore size ranges of scaffolds for stem cell differentiation into specific cell types. Thus, if a scaffold with a pore size gradient can be readily fabricated, it may become a powerful tool for the basic study of the interactions between cells or tissues and scaffolds with different pore sizes. This is attributed to the fact that no individual scaffolds with different pore sizes are needed and the effect of pore size can be effectively examined using one gradient scaffold. In addition, to fabricate the scaffolds with different pore sizes separately may induce experimental artifacts due to the low repeatability of pore size distribution. In this study, we fabricated polycaprolactone (PCL) cylindrical scaffolds with a pore size gradient (i. e., gradually increasing pore size along the longitudinal direction) by a modified centrifugation method. The scaffold was simply obtained by the centrifugation of a cylindrical mold containing fibril-like PCLs and the following fibril bonding by heat treatment. The pore size ranges of the scaffold could be controlled by adjusting the centrifugal speed. To investigate the chondrogenic differentiation in terms of pore sizes in the scaffold using the pore size gradient PCL cylindrical scaffolds, the ASC was chosen as a model stem cell because of its abundance and accessibility, even though it was reported to have a lower chondrogenic differentiation capacity than the BMSC. The chondrogenic differentiation behaviors in the pore size gradient scaffold were assessed by DNA and GAG contents, gene expression, and histological examinations.
Experimental Section Materials. PCL (Mw 42500; Aldrich, Milwaukee, WI), tetraglycol (glycofurol; Sigma, St. Louis, MO), and Pluronic F127 (EG99PG65EG99,
10.1021/bm100199m 2010 American Chemical Society Published on Web 07/20/2010
Differentiation of Adipose Stem Cells
Biomacromolecules, Vol. 11, No. 8, 2010
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Figure 1. Schematic diagram showing the fabrication process of pore size gradient PCL scaffold by a modified centrifugation method.
Mw 12500; BASF, Parsippany, NJ) were used to fabricate pore size gradient PCL scaffolds. Transforming growth factor β2 (TGF-β2) and bone morphogenetic protein 7 (BMP-7) were selected as growth factors to enhance the chondrogenic differentiation of ASCs20 and were purchased from R&D Systems (Minneapolis, MN). All other chemicals were of analytical grade and were used as received. Water was purified (
