Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold

Recent studies have shown that liposuction aspirates from rat, rabbit, mouse, and human sources contain pluripotent adipose tissue-derived stromal cells (ASCs) that can differentiate into various mesodermal cell types, including osteoblasts, myoblasts, chondroblasts, and preadipocytes. To develop a research model for autologous bone tissue engineering, we isolated ASCs from human liposuction aspirates (hASCs) and induced their osteogenic differentiation in three-dimensional poly(dl-lactic-co-glycolic acid) (PLGA) scaffolds. Human liposuction aspirates were proteolytically digested and centrifuged to obtain hASCs. After primary culture in control media and expansion to three passages, the cells were seeded in two-dimensional plates or three-dimensional PLGA scaffolds and cultured in osteogenic media for 4 weeks. In two-dimensional culture, osteogenesis was assessed by RT-PCR analysis of the osteogenic-specific bone sialoprotein mRNA, by alkaline phosphatase staining, and by von Kossa staining. In three-dimensional culture, osteogenesis was assessed by von Kossa and alizarine red S staining at 1, 2, and 4 weeks following osteogenic induction. hASCs incubated in two-dimensional osteogenic media stained positively for alkaline phosphatase and with von Kossa stain after 2 weeks of differentiation. Expression of the osteogenesis-specific bone sialoprotein gene was detected by RT-PCR after 2 weeks of differentiation. PLGA scaffolds seeded with hASCs showed multiple calcified extracellular matrix nodules by von Kossa and alizarine red S staining after 2 weeks of differentiation. In conclusion, the authors identified an osteogenic potential of hASCs and demonstrated osteogenic differentiation of hASCs into an osteogenic lineage in three-dimensional PLGA scaffolds.