EXPRESSION OF BONE ASSOCIATED MARKERS BY TOOTH ROOT LINING CELLS, IN-SITU AND IN-VITRO

Periodontal disease is marked by inflammation and subsequent loss and/ or damage to tooth-supporting tissues including bone, cementum, and pe riodontal ligament. A key tissue in the initial process of periodontal development as well as regeneration following periodontal disease is cementum. Research efforts aimed toward understanding mechanisms invol ved in periodontal development and regeneration, and in particular the formation of root cementum, have,been hampered by an inability to iso late and culture cells involved in cementum production (i.e., cementob lasts). Much has been learned regarding the processes and mechanisms i nvolved in bone formation and function from experiments using bone cel l cultures. Therefore, the purpose of this study was to develop a stra tegy whereby cementoblasts could be isolated, cultured, and characteri zed. As a first step, using in situ hybridization, we determined the t imed and spatial expression of mineral-associated proteins during firs t molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteoc alcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the too th root surface-cementoblasts-with high levels of expression at day 41 . Importantly, at this time point BSP, OPN, and OCN mRNAs were not exp ressed throughout the periodontal ligament. These findings provided us with markers selective to root-lining cells, or cementoblasts, in sit u, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, e nzymatic digestion was used, similar to what are now considered classi cal techniques for isolation of osteoblasts. To determine whether cell s in vitro contained root-lining cells and cementoblasts, cultured cel ls were analyzed for expression of mineral-associated proteins. Cells within this heterogeneous primary population expressed type I collagen , BSP, OPN, and OCN as determined by in situ hybridization. In contras t, cells within this population did not express dentin sialoprotein, a n odontoblast-specific protein. These procedures have provided a means to obtain root-lining cells in vitro that can now be cloned and used for studies directed at determining the properties of root-lining cell s, or cementoblasts, in vitro. (C) 1997 by Elsevier Science Inc.