NORMAL HUMAN CEMENTUM-DERIVED CELLS - ISOLATION, CLONAL EXPANSION, AND IN-VITRO AND IN-VIVO CHARACTERIZATION

Cultures of primary human cementum-derived cells (HCDCs) were establis hed from healthy premolar teeth extracted for orthodontic reasons. Cem entum was manually dissected, fragmented, and digested twice with coll agenase. Following a thorough wash to remove liberated cells, the rema ining cementum fragments were plated in Dulbecco's modified Eagle's me dium/F12 medium containing 10% fetal bovine serum. Discrete colonies t hat contained cells exhibiting fibroblast-like morphology were visible after 14-21 days of culture. When the colonies became sufficiently la rge, cells from individual colonies were isolated and subcultured. Cem entum-derived cells exhibited low levels or no alkaline phosphatase ac tivity and mineralized in vitro to a lesser degree than human periodon tal ligament (PDL) cells and human bone marrow stromal cell (BMSC) cul tures. To study differentiation capacities of HCDCs, cells were attach ed to hydroxyapatite/tricalcium phosphate ceramic and transplanted sub cutaneously into immunodeficient mice. The transplants were harvested 3, 6, and 8 weeks after transplantation and evaluated histologically, In human BMSC transplants, new bone tissue was formed with a prominent osteoblastic layer and osteocytes embedded in mineralized bone matrix . No osseous tissue was formed by PDL cells. Of six single colony-deri ved strains of HCDCs tested, three formed a bone-like tissue that feat ured osteocyte/cementocyte-like cells embedded within a mineralized ma trix and which was lined with a layer of cells, although they were som ewhat more elongated than osteoblasts, These results show that cells f rom normal human cementum can be isolated and expanded in vitro. Furth ermore, these cells are capable of differentiating and forming mineral ized tissue when transplanted into immunodeficient mice.