Gary E. Wise

Mechanisms of Tooth Eruption and Orthodontic Tooth Movement

Teeth move through alveolar bone, whether through the normal process of tooth eruption or by strains generated by orthodontic appliances. Both eruption and orthodontics accomplish this feat through similar fundamental biological processes, osteoclastogenesis and osteogenesis, but there are differences that make their mechanisms unique. A better appreciation of the molecular and cellular events that regulate osteoclastogenesis and osteogenesis in eruption and orthodontics is not only central to our understanding of how these processes occur, but also is needed for ultimate development of the means to control them. Possible future studies in these areas are also discussed, with particular emphasis on translation of fundamental knowledge to improve dental treatments.

CELLULAR, MOLECULAR, AND GENETIC DETERMINANTS OF TOOTH ERUPTION

Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Mononuclear cells (osteoclast precursors) must be recruited into the dental follicle prior to the onset of eruption. These cells, in turn, fuse to form osteoclasts that resorb alveolar bone, forming an eruption pathway for the tooth to exit its bony crypt. Some of the molecules possibly involved in the signaling cascades of eruption have been proposed in studies from null mice, osteopetrotic rodents, injections of putative eruption molecules, and cultured dental follicle cells. In particular, recruitment of the mononuclear cells to the follicle may require colony-stimulating factor-one (CSF-1) and/or monocyte chemotactic protein-1 (MCP-1). Osteoclastogenesis is needed for the bone resorption and may involve inhibition of osteoprotegerin transcription and synthesis in the follicle, as well as enhancement of receptor activator of NF kappa B ligand (RANKL), in the adjacent alveolar bone and/or in the follicle. Paracrine signaling by parathyroid-hormone-related protein and interleukin -1 alpha, produced in the stellate reticulum adjacent to the follicle, may also play a role in regulating eruption. Osteoblasts might also influence the process of eruption, the most important physiologic role likely being at the eruptive site, in the formation of osteoclasts through signaling via the RANKL/OPG pathway. Evidence thus far supports a role for an osteoblast-specific transcription factor, Cbfa1 (Runx2), in molecular events that regulate tooth eruption. Cbfa1 is also expressed at high levels by the dental follicle cells. This review concludes with a discussion of the several human conditions that result in a failure of or delay in tooth eruption.

Differentiation of Stem Cells in the Dental Follicle

The dental follicle (DF) differentiates into the periodontal ligament. In addition, it may be the precursor of other cells of the periodontium, including osteoblasts and cementoblasts. We hypothesized that stem cells may be present in the DF and be capable of differentiating into cells of the periodontium. Stem cells were identified in the DF of the rat first mandibular molar by Hoechst staining, alkaline phosphatase staining, and expression of side-population stem cell markers. These cells were shown to be able to differentiate into osteoblasts/cementoblasts, adipocytes, and neurons. Treating the DF cell population with doxorubicin, followed by incubation in an adipogenesis medium, suggested that the adipocytes originated from stem cells. Thus, a possibly puripotent stem cell population is present in the rat DF.

Changes in the Tartrate-resistant Acid Phosphatase Cell Population in Dental Follicles and Bony Crypts of Rat Molars during Tooth Eruption

It was the aim of this study to determine the cellular changes that occur in the enamel organ, dental follicle, and surrounding bony crypt of the rat molar prior to and during tooth eruption. By use of light microscope histochemistry to detect cells containing tartrate-resistant acid phosphatase (TRAP), it was seen that TRAP-positive mononuclear cells were present in the dental follicle prior to the onset of eruption (e.g., three days postnatal age) and then declined in number during eruption. Concurrently, TRAP-positive osteoclasts were initially present in large numbers on the surface of the bony crypt surrounding the molars (three days postnatal age) and then declined in number as eruption progressed. Electron microscopy confirmed that these were mononuclear cells and osteoclasts. The results suggest that the mononuclear cells are either precursors of the osteoclasts or perhaps release cytokines that affect osteoclast formation or activity. Staining for alkaline phosphatase (ALP) activity indicated that at an early postnatal age (secretory stage of amelogenesis), ALP was detected only in the stratum intermedium of the enamel organ, whereas at a later age (maturation phase of amelogenesis), it was present only in the ameloblasts. These results, combined with a survey of the literature, strongly suggest that ALP moves from the base of the enamel organ to the enamel itself over a period of time ranging from pre- to post-eruption. Rat molars are teeth of limited eruption, and the cellular events that occur in eruption appear comparable with what is seen in dog and human dentition, especially in terms of the cellular events seen in the dental follicle prior to and during eruption. Thus, because rat molars are often more amenable to experimental protocol, they may be a suitable choice for answering questions pertaining to tooth eruption in the dog and in humans.

Cellular and molecular basis of tooth eruption.

OBJECTIVES Tooth eruption requires the presence of a dental follicle (DF), alveolar bone resorption for an eruption pathway, and alveolar bone formation at the base of the bony crypt. The objectives of our investigations have been to determine how the DF regulates both the osteoclastogenesis and osteogenesis needed for eruption. MATERIAL AND METHODS Multiple experimental methods have been employed. RESULTS The DF regulates osteoclastogenesis and osteogenesis by regulating the expression of critical genes in both a chronological and spatial fashion. In the rat 1st mandibular molar there is a major burst of osteoclastogenesis at day 3 postnatally and a minor burst at day 10. At day 3, the DF maximally expresses colony-stimulating factor-1 (CSF-1) to down-regulate the expression of osteoprotegerin (OPG) such that osteoclastogenesis can occur. At day 10, the minor burst of osteoclastogenesis is promoted by upregulation of vascular endothelial growth factor (VEGF) and RANKL in the DF. Spatially, the bone resorption is in the coronal portion of the bony crypt and genes such as RANKL are expressed more in the coronal region of the DF than in its basal one-half. For osteogenesis, bone formation begins at day 3 at the base of the bony crypt and maximal growth is at days 9-14. Osteo-inductive genes such as bone morphogenetic protein-2 (BMP-2) appear to promote this and are expressed more in the basal half of the DF than in the coronal. Conclusion - The osteoclastogenesis and osteogenesis needed for eruption are regulated by differential gene expression in the DF both chronologically and spatially.

Transcription and Translation of CSF-1 in the Dental Follicle

The dental follicle, a loose connective tissue sac which surrounds the unerupted tooth, is required for eruption to occur. Injection of colony-stimulating factor-1 (CSF-1) will accelerate molar eruption in rats, as well as stimulate tooth eruption in osteopetrotic rats. Utilizing in situ hybridization and reverse- transcription polymerase chain-reaction techniques, we show here that CSF-1 mRNA is present in vivo in the dental follicle of the first mandibular molar of the rat. Analysis of the molars from day 0 through day 10 post-natally demonstrates that the maximal expression of CSF-1 mRNA is at day 3 post-natally. Immunostaining also reveals that the CSF-1 mRNA is translated, with immunostaining for the CSF-1 itself, being heavy in early post-natal days and absent by day 9 post-natally. In view of the fact that there is a maximal influx of mononuclear cells (monocytes) into the dental follicle at day 3 post-natally-an influx which increases the numbers of osteoclasts needed to form a tooth eruption pathway-it is probable that the maximal expression of CSF-1 mRNA by day 3 post-natally contributes to this monocyte influx. Thus, this study establishes a relationship among a molecule (CSF-1), cell (monocyte), and tissue (dental follicle) that appear to play a major role in tooth eruption.

Osteoprotegerin and Osteoclast Differentiation Factor in Tooth Eruption

A critical cellular event in tooth eruption is the formation of osteoclasts that are needed for bone resorption to form an eruption pathway. To analyze molecular regulation of osteoclast formation and activation, we examined the expression of osteoprotegerin (OPG), an inhibitor of osteoclast formation. In vivo, the gene expression of OPG is reduced in the dental follicle of the first mandibular molar of the rat at day 3 post-natally and in the mouse at day 5. This correlates with the days of maximal mononuclear cell influx and osteoclast numbers in the rat and mouse. Thus, inhibition of OPG gene expression on these days might allow osteoclasts to be formed and/or activated. In vitro studies demonstrated that both colony-stimulating factor-1 and parathyroid hormone-related protein reduced OPG gene expression in follicle cells, suggesting that these are candidate molecules for the in vivo inhibition of OPG expression. Osteoclast differentiation factor (ODF) immunolocalizes to the alveolar bone stromal cells adjacent to the follicle, whereby it might act to stimulate fusion of the mononuclear cells in the follicle.

Inhibition of Tooth Eruption in the Rat by a Bisphosphonate

Studies of osteopetrotic rodents suggest that localized alveolar bone resorption must occur if the tooth is to To test this hypothesis directly, we injected postnatal rats with pamidronate, a bisphosphonate that reduces bone resorption by osteoclasts, The results of these experiments demonstrate that this bisphosphonate inhibits the time of tooth of both rat molars and incisors. Pamidronate does not inhibit the gene expression of the putative tooth eruption molecules, colony-stimulating factor-1 and c-fos, both of which are expressed in the dental follicle, the tissue that is required for eruption to occur. Pamidronate does increase the size of the osteoclasts, including an increase in the number of nuclei, suggesting that the precursor mononuclear cells can still fuse to form osteoclasts despite the reduced ability of the osteoclasts to resorb bone, Thus, we report the discovery of an agent that inhibits tooth eruption and also show that tooth eruption requires alveolar bone resorption.

Concise Review: The Molecular Biology of Initiation of Tooth Eruption

T he molecular biology revolution of the latter part of this century has affected all areas of biomedical research. Thus, in dental research, the tools of this revolution can now be used to establish what the molecular signals are that initiate tooth eruption. Answering the question of what molecule(s) signal a semi-hard object, the tooth, to escape from its bony crypt is not only an intriguing molecular and developmental biology question but also a question of clinical significance. Knowing what molecules are required to initiate normal tooth eruption would be of enormous benefit in devising a molecular approach to stimulating eruption of impacted teeth. Equally significant, knowledge of the role of these molecules in bone resorption will be acquired because bone resorption is required for eruption (e.g., see Sundquist and Marks, 1994; Marks et al., 1995). Before one can establish the molecular basis of tooth eruption, one must first know what cells and tissues are required for eruption-i.e., upon what tissue(s) are the signaling molecule(s) required to act? In what chronological period of development does the signaling occur? Thanks to the pioneering experiments of Marks and Cahill, it was established that, in teeth of limited eruption, a tissue required for eruption is the dental follicle, a loose connective tissue sac that surrounds the tooth prior to eruption. Their studies showed that surgical removal of the follicle prevents eruption (Cahill and Marks, 1980), whereas leaving the follicle intact but substituting an inert object for the tooth results in eruption of the inert object (Marks and Cahill, 1984). At the cellular level, there is an influx of mononuclear cells (monocytes) into the dental follicle, beginning at 14 weeks post-natally in the dog 3rd premolar, and this influx peaks at 16 weeks, which is the onset of

Sodium dodecyl sulfate-gel electrophoresis: Staining of polypeptides using heavy metal salts

Water-soluble salts of several heavy metals were examined for their ability to stain polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Brief gel exposure (5 min or less) to cobaltous acetate or chlorides of copper, nickel, and zinc produced negatively stained protein patterns that were qualitatively indistinguishable from those of parallel gels stained with Coomassie blue R-250. Protein patterns could be visualized less than 1 min after treatment of gels with zinc chloride; the threshold of detection was estimated at about 10-12 ng protein on standard-size slab gels. Test samples including human erythrocyte membranes, sialoglycoprotein (glycophorin) extracts, and commercial molecular weight protein standards were used to establish the scope of these stains. Protein patterns visualized by the heavy metal salts were compared and contrasted with profiles seen with three widely used silver stains. Proteins from gels treated with copper or zinc chloride could be easily recovered by simple diffusion; this makes feasible both analytical and preparative electrophoretic applications of the staining procedure. A mechanism is proposed to explain the observed protein staining by heavy metal salts.