The effect of hydrostatic pressure on three-dimensional chondroinduction of human adipose-derived stem cells.
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.


CONCLUSIONS
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.
Ogawa
Rei
R
Division of Plastic Surgery, Department of Surgery, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA. r.ogawa@nms.ac.jp
Mizuno
Shuichi
S
Murphy
George F
GF
Orgill
Dennis P
DP
D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D002356:Cartilage / Q000166:cytology* / Q000378:metabolism; D002454:Cell Differentiation; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D006874:Hydrostatic Pressure; D007150:Immunohistochemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds / Q000737:chemistry*
D003094:Collagen