Osteogenic potentiation of human adipose-derived stem cells in a 3-dimensional matrix

Adipose-derived stem cells (ADSCs) hold promise for use in tissue engineering. Despite growing enthusiasm for use of ADSCs, there is limited research that has examined their behavior in different in vitro and in vivo systems. The purpose of our study was to evaluate the effect of the extracellular matrix structure and composition on osteogenic differentiation by comparing the osteogenic marker expression of ADSCs grown under 2-dimensional or 3-dimensional cell culture conditions. Group 1 (2-D) included ADSCs raised under conventional cell culture conditions (cells in a 2-D monolayer configuration) (n = 24), and group 2 (3-dimensional) included ADSCs seeded in a collagen gel (cells within a 3-dimensional, biologically active environment) (n = 24). Comparison of ADSC behavior between the 2 groups was analyzed during a 14-day time frame. Osteogenic marker expression (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, Collagen 1, and JNK2) was quantified by real-time PCR, and histologic analysis was performed. Histologically, group I (2-D) showed cell spreading and deposition of a calcified extracellular matrix. Group 2 (3-dimensional) assumed a disorganized state in the collagen gel, with extension of pseudopodia throughout the matrix. Expression of CBFA-1 was up-regulated immediately in both groups. However, cells in group 2 (3-dimensional) had a more rapid and greater overall expression compared with cells in group 1 (2-D) (250-fold greater at 4 days). At day 14, cells in group 2 (3-dimensional) showed greater expression of all other osteogenic markers than cells in group 1 (2-D) (2.3-fold greater expression of alkaline phosphatase [P < 0.05]. 8.4-fold greater expression of osteonectin [P < 0.05], 6.4-fold greater expression of osteopontin [P < 0.05], 2.9-fold greater expression of collagen I [P < 0.05], and 2.5-fold greater expression of JNK2 [P < 0.05]). Our data showed there was a progressive stimulatory effect on ADSCs with regard to osteogenesis when cultured in a 3-dimensional gel compared with a 2-D monolayer.