Periodontal ligament cells play a crucial role in the regeneration of periodontal tissues and an undifferentiated mesenchymal cell subset is thought to exist within this population. The aim of this study was to assess the osteogenic differentiation potential of human periodontal ligament fibroblasts (hPDLFs) in three dimensional (3D)osteogenic culture environment following encapsulation in chitosan-hydroxyapatite (C/HA) microspheres with the size range of 350-450 mu m. Human PDLF cultures were established and three experimental groups were formed: (i) two-dimensional (2D)-culture as single cell monolayer, (ii) 3D-static culture of C/HA encapsulated hPDLFs, and (iii) 3D-dynamic culture of C/HA encapsulated hPDLFs in a rotating wall vessel bioreactor. The cells were cultured in standard culture medium supplemented with P-glycerophosphate, dexamethasone, and ascorbic acid. After 21 days, immunohistochemistry was performed using antibodies against osteonectin, osteopontin, bone-sialoprotein, and osteocalcin as osteogenic differentiation markers. Phase-contrast and scanning electron microscopy observations were used for histological and morphological evaluation. The combined effects of osteoinductive medium and HA-containing composite microsphere material on encapsulated hPDLFs resulted in the transformation of a considerable portion of the cells into osteoblastic lineage at the end of the experiments. Results demonstrate the ability of hPDLFs to undergo osteogenic differentiation upon induction in vitro, both under 2D and 31) culture conditions. C/HA microspheres in microgravity bioreactor may serve as a suitable 31) environment to support the osteogenic differentiation of human PDLFs, in vitro. (c) 2007 Wiley Periodicals, Inc.