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. 2009 Oct;15(10):2937-45.
doi: 10.1089/ten.TEA.2008.0672.

The Effect of Hydrostatic Pressure on Three-Dimensional Chondroinduction of Human Adipose-Derived Stem Cells

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Free PMC article

The Effect of Hydrostatic Pressure on Three-Dimensional Chondroinduction of Human Adipose-Derived Stem Cells

Rei Ogawa et al. Tissue Eng Part A. .
Free PMC article

Abstract

Background: The optimal production of three-dimensional cartilage in vitro requires both inductive factors and specified culture conditions (e.g., hydrostatic pressure [HP], gas concentration, and nutrient supply) to promote cell viability and maintain phenotype. In this study, we optimized the conditions for human cartilage induction using human adipose-derived stem cells (ASCs), collagen scaffolds, and cyclic HP treatment.

Methods: Human ASCs underwent primary culture and three passages before being seeded into collagen scaffolds. These constructs were incubated for 1 week in an automated bioreactor using cyclic HP at 0-0.5 MPa, 0.5 Hz, and compared to constructs exposed to atmospheric pressure. In both groups, chondrogenic differentiation medium including transforming growth factor-beta1 was employed. One, 2, 3, and 4 weeks after incubation, the cell constructs were harvested for histological, immunohistochemical, and gene expression evaluation.

Results: In histological and immunohistochemical analyzes, pericellular and extracellular metachromatic matrix was observed in both groups and increased over 4 weeks, but accumulated at a higher rate in the HP group. Cell number was maintained in the HP group over 4 weeks but decreased after 2 weeks in the atmospheric pressure group. Chondrogenic-specific gene expression of type II and X collagen, aggrecan, and SRY-box9 was increased in the HP group especially after 2 weeks.

Conclusion: Our results demonstrate chondrogenic differentiation of ASCs in a three-dimensional collagen scaffolds with treatment of a cyclic HP. Cyclic HP was effective in enhancing accumulation of extracellular matrix and expression of genes indicative of chondrogenic differentiation.

Figures

FIG. 1.
FIG. 1.
Stem sell seeding into the collagen scaffold. Cultured passage 3 adipose–derived stem cells (ASCs) were harvested and resuspended with neutralized type I collagen solution, and each 50 μL of cell suspension (1 × 106 cells) was absorbed with each collagen sponge scaffold and incubated at 37°C for gelation. Color images available online at www.liebertonline.com/ten.
FIG. 2.
FIG. 2.
Semiquantitive histological analysis. Toluidine blue-hematoxylin–stained sections were analyzed for cell proliferation quantification on both surface and middle zone. High-power digital images of sections were used to measure the total number of hematoxylin-positive nuclei. The degree of proliferation was quantified over the entire sponge using three fields in each zone at 40 magnification. SZ, surface zone; MZ, middle zone. Color images available online at www.liebertonline.com/ten.
FIG. 3.
FIG. 3.
Histological analysis. Accumulation of pericellular and extracellular metachromatic matrix that stained with toluidine blue within collagen scaffolds was observed in both groups and increased over 4 weeks. Accumulation of the matrix in the HP group was much greater than AP groups especially after 2 weeks. Immunohistochemical evaluation revealed that expression of type II and keratan sulfate of both groups increased with time, and HP groups showed greater expression than AP groups at each time point. HP, hydrostatic pressure treated groups; AP, atmospheric pressure control groups. Color images available online at www.liebertonline.com/ten.
FIG. 4.
FIG. 4.
Semiquantitative analysis of histology. Cell number in AP groups achieved a peak at 2 weeks of incubation and decreased after peaking on both middle and surface zone of sponges. On the other hand, that of HP groups increased gradually on middle zone, and peaked at a week after incubation and kept their number in the entire course on surface zone. MZ, middle zone; SZ, surface zone; HPF, high-power field (40 × images); AP, atmospheric pressure group; HP, hydrostatic pressure group.
FIG. 5.
FIG. 5.
Real-time RT-PCR. Three sponges in each group at each time points were analyzed by real-time RT-PCR. Chondrogenic-specific gene expression of type II and X collagen, ACAN, and Sox9 were observed on both groups. However, there was significant differences between HP and AP groups after 2 weeks of culture on ACAN and Sox9, after 3 weeks of culture on type X collagen, and at 2 and 4 weeks of time point on type II collagen. ACAN, aggrecan; Sox9, SRY-box9; COL2A1, type II collagen α-1; COL10A1, type X collagen α-1; AP, atmospheric pressure group; HP, hydrostatic pressure group.

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