R. L. MacNeil

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 periodontal 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 involved in periodontal development and regeneration, and in particular the formation of root cementum, have been hampered by an inability to isolate and culture cells involved in cementum production (i.e., cementoblasts). Much has been learned regarding the processes and mechanisms involved in bone formation and function from experiments using bone cell cultures. Therefore, the purpose of this study was to develop a strategy whereby cementoblasts could be isolated, cultured, and characterized. As a first step, using in situ hybridization, we determined the timed and spatial expression of mineral-associated proteins during first molar root development in CD-1 mice. These proteins included dentin sialoprotein (DSP), osteopontin (OPN), bone sialoprotein (BSP), osteocalcin (OCN), and type I collagen. During root development in mice BSP, OPN, and OCN mRNAs were expressed selectively by cells lining the tooth root surface--cementoblasts--with high levels of expression at day 41. Importantly, at this time point BSP, OPN, and OCN mRNAs were not expressed throughout the periodontal ligament. These findings provided us with markers selective to root-lining cells, or cementoblasts, in situ, and established the time (day 41) for isolating cells for in vitro studies. To isolate cells from tissues adherent to the root surface, enzymatic digestion was used, similar to what are now considered classical techniques for isolation of osteoblasts. To determine whether cells in vitro contained root-lining cells and cementoblasts, cultured cells 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 contrast, cells within this population did not express dentin sialoprotein, an 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 cells, or cementoblasts, in vitro.

Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis.

Adhesion molecules and their cell membrane receptors are known to play important regulatory roles in cell differentiation. Consequently, the following experiments were conducted to determine the role of two adhesion molecules, bone sialoprotein (BSP) and osteopontin (OPN) in tooth root formation. Developing murine molar tooth germs at sequential stages of development (developmental days 21-42) were analyzed using immunohistochemical and in situ hybridization techniques. While BSP was localized to alveolar bone and odontoblasts early in development, BSP was distinctly localized to the cemental root surface at latter periods coincident with the initiation of root formation and cementogenesis. Conversely, OPN was distributed in a nonspecific fashion throughout the PDL and the eruption pathway of the forming tooth. In situ hybridization confirmed that cells lining the root surface express BSP. The fact that BSP is specifically localized to the cemental surface suggests that this protein is involved in cementoblast differentiation and/or early mineralization of the cementum matrix. Localization of OPN to non-mineralized tissues further suggests that OPN functions as an inhibitor of mineralization during periodontal ligament formation. These findings collectively suggest that BSP and OPN are intimately involved in the sequence of cellular and molecular events accompanying cementogenesis.

Development of the Murine Periodontium. I. Role of Basement Membrane in Formation of a Mineralized Tissue on the Developing Root Dentin Surface

The presence of basement membrane components on the forming root surface suggests a role for this structure during cementoblast differentiation. The purpose of this study was to investigate the role of root-associated basement membrane (RBM) in murine cementogenesis using tissue separation and recombination techniques. Root dentin specimens, with or without RBM, were combined with dental sac tissue, cultured for 2 weeks, harvested, and examined by light, immunofluorescence, and electron microscopy. Recombinations in which RBM was preserved on the root dentin surface were characterized by formation of an adherent mineralized tissue resembling acellular cementum; recombinations in which RBM was excluded were characterized by formation of mineralized tissue which did not adhere to the root dentin surface. These results suggest that formation of an adherent mineralized tissue on the developing root dentin surface is dependent upon the presence of indigenous basement membrane components.

Expression of type I and XII collagen during development of the periodontal ligament in the mouse

The purpose (of this study) was to determine the temporal and spatial pattern of type XII collagen expression during murine tooth/root development. Using in situ hybridization techniques, expression of type XII collagen was compared with that of type I collagen, the most abundant collagen in periodontal tissues. Mouse first mandibular molars were examined at the following developmental periods: pre-root formation, early root formation, initial alignment of the periodontal ligament (PDL) fibres, and PDL maturation as the tooth erupted and attained occlusal function. Transcripts for type I collagen were identified in bone cells and odontoblasts at all times but not in the dental follicle before root formation. As root formation progressed, type I collagen expression became apparent within cells of the dental follicle and forming PDL. During early stages of tooth development, signal for type XII collagen was not observed in any cells/tissues. Type XII collagen expression was first detected in the dental follicle/PDL region during tooth eruption and increased in the PDL as the molar tooth erupted into the mouth and achieved occlusal contact. These findings suggest that type XII expression is timed with the alignment and organization of PDL fibres and is limited in tooth development to cells within the periodontal ligament.

Localization and expression of osteopontin in mineralized and nonmineralized tissues of the periodontium

To summarize results from various studies focusing on determining the expression/localization of BSP and OPN during tooth root development, there is general agreement that OPN is expressed/localized to the root surface during cementogenesis and is also seen throughout the PDL region. The expression/localization of OPN to odontoblasts and its role in dentinogenesis is less apparent. Recent studies directed at establishing odontoblast cell lines should help to resolve this conflict. Studies on BSP expression during tooth root formation indicate a very precise expression and localization of this molecule during cementogenesis indicating that this molecule may play an important role in the formation of this mineralized tissue. However, as with OPN, the expression of BSP and its role in dentin formation is not clearly defined.

Agents with Periodontal Regenerative Potential Regulate Cell-mediated Collagen Lattice Contraction in vitro

A variety of pharmaceutical agents has been proposed for use in periodontal therapy to inhibit loss of alveolar bone and to promote regeneration of tissues lost to disease. The purpose of this study was to determine the effects of such agents on periodontal cell-mediated gel contraction, an in vitro process considered representative of wound contraction and remodeling in vivo. Human gingival fibroblasts were cultured in a type I collagen lattice, and contraction was quantified by means of a computer-assisted video imaging system. Cell-gel combinations were prepared with cells both pre-exposed and non-exposed to agents; non-anchored cell-gels were then incubated with agents for various time periods. Agents tested included Demecolcine (an inhibitor of cytoskeletal contraction), growth factors (i.e., TGF-β1, PDGF, and IGF-1), and non-steroidal anti-inflammatory drugs (NSAIDs) (indomethacin, ibuprofen, naproxen, and flurbiprofen). While Demecolcine inhibited gel contraction, TGF-β1 (1-20 ng/mL), PDGF (100 ng/mL), IGF-1 (1000 ng/mL), and [PDGF + IGF], all of which have been reported to enhance wound healing in vivo, promoted lattice contraction in this system. In contrast, NSAIDs inhibited cell-gel contraction. Ethanol, used to solubilize two specific NSAIDs, also inhibited cell proliferation and gel contractile ability, even at very low concentrations. These findings indicate that periodontal cells respond differently to various agents in vitro and may be adversely affected by alcohol. Furthermore, the results of this study suggest that the cell-lattice contraction system holds potential as a method for screening agents for positive or negative effects on cell activity.

Models for the study of cementogenesis.

Cementum is a mineralized tissue that acts to connect the periodontal ligament to the tooth root surface. Its composition is very much like bone, being comprised mainly of type I collagen, inorganic mineral and noncollagenous proteins, however the origin of the cells and factors necessary for cementum formation have yet to be elucidated. Our laboratory has focused on the role that adhesion molecules, and their cell surface receptors, play in the formation of cementum and tooth root. In order to study this, we used a mouse molar as a model system. This system enabled us to study the formation of four distinct mineralized tissues; bone, cementum, dentin and enamel at various stages of their development. For these studies, we initiated experiments to examine potential cementoblast progenitor cells, in vitro. As a first step, we show that dental papilla and dental follicle cells, n vitro, obtained from molar tissues at day 21 of development, induce mineralized nodules, in vitro. In addition, we obtained tissues from mice where defects in root development may exist and determined bone sialoprotein (BSP) protein expression, a mineralized tissue specific adhesion molecule, in such tissues. As discussed here, we found that osteopetrotic (op/op) mice have delayed and/or defective root development and BSP does not localize in the dental tissues, at day 33 of development. In addition, dentin formation was defective and odontoblasts appeared immature, based on morphological examination. In contrast, the day 33 control molars demonstrated positive staining for BSP localized to root cementum, with normal formation of dentin.