Hydrostatic Pressure-Driven Three-Dimensional Cartilage Induction Using Human Adipose-Derived Stem Cells and Collagen Gels

Background: The chondrogenic potential of adipose-derived stem cells (ASCs) has been previously demonstrated, although several reports have indicated that ASCs produce less cartilage-specific matrix than bone marrow-derived mesenchymal stem cells. In this study, we intended to improve chondrogenic phenotypes of ASCs using hydrostatic pressure (HP), without utilizing any growth factors other than the transforming growth factor-β1. Methods: Human ASCs (CD13 + , 44 + , 90 + , 14 − , 31 − , 34 − ) were harvested and cultured. After three passages, the cells were suspended in 0.3% neutralized collagen type I solution and injected into semipermeable membrane tubes, from which 66 pouches were constructed. After a day of incubation, the 66 pouches were divided into three groups. Group HP1: Pouches were incubated for 1 week with treatment of cyclic HP at 0–0.5 MPa (4.93 atm), 0.5 Hz, with a medium replenishment rate of 0.1 mL/min at 37°C, 3% O 2 , and 5% CO 2 in air using a bioprocessor. This was followed by 3 weeks with no HP and without pouches. Group HP2: Pouches were incubated for the first and third week (2 total weeks) with the same condition of Group HP1. No HP was applied in the second and fourth week. Group AP: Pouches with one end opened were incubated without HP. We evaluated the cell constructs histologically and immunohistochemically, as well as for specific gene expression. Results: Accumulation of the matrix in the HP1 and HP2 groups was much denser than AP groups, particularly after 2 weeks. Cell numbers in the HP groups increased gradually in the middle zone and peaked at 1 week after incubation, maintaining their numbers for the entire course on the surface layer of the construct. In the genomic study results, COL 2A1 , COL 10A1 , ACAN , SOX9 , MMP3 , and MMP13 were upregulated and COL 1A1 , ITGB1 , and PCNA were downregulated by HP. There were no significant differences between HP1 and HP2 gene expression. Conclusion: It was suggested that HP is especially beneficial in the early stage of chondrogenesis of ASCs. Moreover, the expression profile of genes related to chondrocyte differentiation/proliferation was significantly enhanced by HP loading compared with the AP control.