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Mechanisms of Tooth Eruption and Orthodontic Tooth Movement

¹ Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA; and
² Department of Orthodontics, School of Dentistry, University of Washington, Seattle, WA 98195, USA

^* corresponding author, gwise{at}vetmed.lsu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION REQUIREMENTS FOR TOOTH ERUPTION... MOLECULAR REGULATION OF... MOLECULAR REGULATION OF... UNDERLYING BIOLOGICAL AND... FUTURE STUDIES CONCLUSIONS REFERENCES

 
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.

KEY WORDS: dental follicle • periodontal ligament • osteoclastogenesis • osteogenesis • RANKL • OPG • CSF-1 • bone remodeling • bone formation • bone resorption

	INTRODUCTION

TOP ABSTRACT INTRODUCTION REQUIREMENTS FOR TOOTH ERUPTION... MOLECULAR REGULATION OF... MOLECULAR REGULATION OF... UNDERLYING BIOLOGICAL AND... FUTURE STUDIES CONCLUSIONS REFERENCES

 
Given the breadth of the two topics, tooth eruption and orthodontic tooth movement, this review will focus more upon what is currently known about their molecular mechanisms, commonalities, and differences, instead of a lengthy historical review. For the latter, readers are referred to other reviews (Cahill et al., 1988; Marks and Schroeder, 1996; Wise et al., 2002; Krishnan and Davidovitch, 2006; Masella and Meister, 2006; Meikle, 2006).

Theories of Tooth Eruption
For the past 70 years, various theories have been presented on the mechanisms of tooth eruption. That numerous theories abound may be due to the enormous success of orthodontics in moving teeth with force application. However, tooth eruption is a fundamental developmental and physiologic process, and force plays a secondary role. Regardless, some of these theories are discussed in the section of this review entitled "Motive Force of Tooth Eruption". Previous reviews in the past 20 years also have considered the various theories of eruption (e.g., see Cahill et al., 1988; Marks and Schroeder, 1996; Wise et al., 2002).

In this review, emphasis will be on the dental follicle and its role in initiating eruption by regulating alveolar bone resorption and alveolar bone formation. This focus emanates from the pioneering work of Sandy Marks, Jr., and Don Cahill, who demonstrated that the dental follicle was required for eruption. Their surgical studies utilizing dog premolars showed that removal of the follicle from the unerupted tooth prevented the tooth from erupting (Cahill and Marks, 1980), whereas leaving the follicle intact and substituting an inert object for the tooth resulted in eruption of the inert object (Marks and Cahill, 1984).

One caveat to remember in this review is that we are focusing on teeth of limited eruption (e.g., human dentition, rodent molars), not on teeth of continuous eruption (e.g., rodent incisors). Different molecules and mechanisms appear to regulate eruption in the two types of teeth. Regarding the molecules, epidermal growth factor accelerates the time of eruption in rodent incisors, but has little effect on the molars (Lin et al., 1992; Cielinski et al., 1995), whereas colony-stimulating factor-1 (CSF-1) accelerates rat molar eruption, but not incisor eruption (Cielinski et al., 1994, 1995). Injection of dexamethasone also has contrasting effects, in that it accelerates incisor eruption, but not molar eruption (Wise et al., 2001).

Pathophysiology of Orthodontic Tooth Movement
Unlike tooth eruption, orthodontic tooth movement is a process that combines both pathologic and physiologic responses to externally applied forces. With the possible exception of tooth drift, which in some ways resembles eruption (King et al., 1991a), orthodontic tooth movement is accompanied by minor reversible injury to the tooth-supporting tissues. Superimposed on this is the physiologic adaptation of alveolar bone to mechanical strains. Therefore, relevant inflammatory mechanisms need to be considered along with skeletal mechanotransduction for a full understanding of orthodontic tooth movement. The evidence for injury and its resolution in orthodontic tooth movement will be considered in this section, and theories for skeletal mechanotransduction will be reviewed separately in a later section. Despite these differences, one similarity in both orthodontic and tooth eruption movement is the requirement for an intervening biologically active soft tissue. In the case of eruption, this is the dental follicle, while in orthodontics it is the periodontal ligament. The data supporting evidence for both of these requirements will be considered in the following section.

The clinical picture of orthodontic tooth movement consists of three phases: an initial and almost instantaneous tooth displacement; delay, where no visible movement occurs; and a period of linear tooth movement. The applied forces create strains in the tooth-supporting tissues that manifest almost immediately and can be roughly categorized as compressive and tensile. In the absence of transducer data directly measuring these strains, various finite element models have been created to describe them. Finite element analyses of the load transfer from the tooth through the periodontal ligament to the alveolar bone must account for the physical properties and morphology of the periodontium. The periodontal ligament is known to be a non-linear visco-elastic material, but orthodontic finite element models often incorporate homogeneous, linear elastic, isotropic, and continuous periodontal ligament properties. Also, adjustments for differences in micromorphology have not been made. Finite element studies that attempt to account for these report that loading of the periodontium cannot be explained in simple terms of compression and tension along the loading direction. Also, tension seems to be far more common than compression (Cattaneo et al., 2005). However, because the pressure-tension terminology is so prevalent in the literature, and it generally can serve as a convenient means to distinguish the different processes accompanying orthodontic tooth movement, it will be used here.

The initiating inflammatory event at compression sites is caused by constriction of the periodontal ligament microvasculature, resulting in a focal necrosis, known by its histological appearance as hyalinization, and compensatory hyperemia in the adjacent periodontal ligament (Murrell et al., 1996) and pulpal vessels (Kvinnsland et al., 1989). These necrotic sites release various chemo-attractants (Lindskog and Lilja, 1983) that draw giant, phagocytic, multi-nucleated, tartrate-resistant acid-phosphatase-positive cells to the periphery of the necrotic periodontal ligament (Brudvik and Rygh, 1994a,b). These cells resorb the necrotic periodontal ligament, as well as the underlying bone and cementum (Fig. 1A). Osteoclasts are recruited from the adjacent marrow spaces (Rody et al., 2001). Until these cells can be recruited and the necrosis removed, tooth movement is impeded, resulting in the clinical manifestation of a delay period. This is followed by deposition of new cementum (Brudvik and Rygh, 1995; Casa et al., 2006), pulpal secondary dentin (Nixon et al., 1993), and bone (King et al., 1991b) in the vicinity of resorption sites.

View larger version (118K): [in this window] [in a new window]   Figure 1. Comparison of compression and tension zones. (A) SEM of resorption cratering in a rat maxillary first molar root adjacent to a compression zone with 40 cN of orthodontic force for 5 days. (B) SEM of a rat maxillary first molar socket. Osteogenesis in response to 40 cN of orthodontic tension for 5 days can be seen as bony spicules extending into the socket from the top of the image.

The release of pro-inflammatory cytokines and lysosomal enzymes that promote tissue resorption at compression sites is well-documented. Prostaglandins, IL-1, IL-6, TNF, and receptor activator of nuclear factor kappa B ligand (RANKL) are all elevated in the periodontal ligament during tooth movement (Yamaguchi and Kasai, 2005). Increases in the lysosomal enzymes, acid phosphatase, tartrate-resistant acid phosphatase (Lilja et al., 1983, 1984; Keeling et al., 1993), and cathepsin B (Yamaguchi et al., 2004) are also localized at compression sites, suggesting that they may play pivotal roles during orthodontic tooth movement in the process of hard- and soft-tissue degradation by increased numbers of macrophage and dendritic-like cells (Vandevska-Radunovic et al., 1997).

Tension sites in orthodontic samples generally have been characterized as being primarily osteogenic, without a significant inflammatory component (Fig. 1B). However, there is evidence that inflammatory responses to tension may be strain-dependent, since tensile strains of low magnitude are anti-inflammatory and induce magnitude-dependent anabolic signals in osteoblast-like periodontal ligament cells, culminating in the regulation of inflammatory gene transcription (Long et al., 2001). In contrast, high tensile strains act as pro-inflammatory stimuli and increase the expression of inflammatory cytokines (Long et al., 2002). This finding has recently been confirmed in a tooth movement model where sites presumed to be in low tensile strain exhibited a marked absence of IL-1 and COX-2, while those presumed to be compressive or having high tensile strains showed up-regulation of IL-1 and COX-2 (Madhavan et al., 2008, submitted). Morphological evidence of periodontal ligament cellular disruption at tension sites in tooth movement also has been described after only 5 min of loading, further suggesting the involvement of an inflammatory mechanism (Orellana et al., 2002; Orellana-Lezcano et al., 2005). Despite this, the mechanism for osteogenesis at tension sites in tooth movement is not well-understood, but reasonable inferences can be made from various mechanotransduction models. These will be discussed in a subsequent section of this review.

One issue that, at first, seems paradoxical is the observation that compression sites in orthodontic tooth movement are primarily resorptive, while the tensile sites are osteogenic. This seems contrary to the bone mechanical usage literature, which describes loaded sites as being osteogenic and unloaded sites as resorptive (Frost, 2004). There are two possible explanations for these differences. First, compression sites clearly have a tissue injury component superimposed on physiological transduction, with the former producing inflammatory products that are primarily resorptive and stimulate cells to remove the injured tissue. Second, resorption at compression sites in tooth movement could be perceived as a result of lowering of the normal strain from the functioning periodontal ligament, while osteogenesis at tension sites could be a reflection of loading of the principal fibers of the periodontal ligament (Melsen, 2001). The latter could also be accompanied by strains in the alveolar process transmitted either through the principal fibers of the periodontal ligament or by direct impingement of the tooth root on the alveolar bone.

Another important distinction between orthodontic tooth movement and eruption may be that there is considerable variation in the response of periodontal ligament tissues to tooth movement. This can be due not only to differences in biomechanical signals, but also to specific host differences, such as diurnal rhythms (Miyoshi et al., 2001), occlusion (Esashika et al., 2003), systemic metabolism (Verna and Melsen, 2003), age (King et al., 1995; Kyomen and Tanne, 1997; Ren et al., 2003), or normal variation in bony trabeculation.

	REQUIREMENTS FOR TOOTH ERUPTION AND ORTHODONTIC TOOTH MOVEMENT

TOP ABSTRACT INTRODUCTION REQUIREMENTS FOR TOOTH ERUPTION... MOLECULAR REGULATION OF... MOLECULAR REGULATION OF... UNDERLYING BIOLOGICAL AND... FUTURE STUDIES CONCLUSIONS REFERENCES

 
There are two fundamental requirements for both tooth eruption and orthodontic tooth movement to occur: (1) Both require soft tissue, intervening between tooth structure and alveolar bone, which plays an important role in regulating the remodeling of adjacent tissues; and (2) both require bone turnover that is temporally and spatially regulated to facilitate specific translocations of teeth through alveolar bone.

The Intervening Biologically Active Soft Tissues
    Dental Follicle for Eruption
With the publication of their seminal papers on the necessity of the dental follicle for tooth eruption, Sandy Marks and Don Cahill not only altered our views of tooth eruption, but also gave us a tissue on which to focus as the molecular regulator of eruption. Interposed between the alveolar bone of the socket and the enamel organ of the unerupted tooth, the dental follicle is a loose connective tissue sac that is ideally positioned to regulate alveolar bone activity (see Fig. 2A). It is highly likely that the reason the dental follicle is needed for eruption is because it initiates and regulates the required osteoclastogenesis and osteogenesis, at least for the intra-osseous phase of eruption leading to tooth emergence. For the supra-osseous phase of eruption, in which the tooth moves to its final occlusal plane, the follicle may play a lesser role, while biomechanical influences may become more important. As demonstrated by Cahill and Marks (1982) in the dog premolar, not until the supra-osseous phase is the dental follicle attached to the alveolar bone and cementum, becoming the periodontal ligament. Similarly, in the first mandibular molar of the rat, the dental follicle becomes the periodontal ligament (Fig. 2B) and does not attach to the alveolar bone and cementum until approximately day 18—the emergence time of the tooth (Wise et al., 2007). In turn, this periodontal ligament could aid in moving the tooth to its occlusal plane during the supra-osseous phase, not unlike what appears to occur in the continuously growing incisors of the rodent (Moxham and Berkovitz, 1974) or rabbit (Berkovitz and Thomas, 1969).

View larger version (84K): [in this window] [in a new window]   Figure 2. Morphology of unerupted tooth vs. erupted tooth. (A) Longitudinal section of a rat first mandibular molar at day 3 post-natally, showing the loose connective tissue sac, the dental follicle (DF), that surrounds the 1st molar (M1). Note that the unerupted tooth is encased in alveolar bone (AB), and that a prominent stellate reticulum is present. (B) Fluorescence micrograph image of a rat maxillary first molar and adjacent tissue with 40 cN of orthodontic force for 5 days. Root (R), periodontal ligament (P), and alveolar bone at compression (C) and tension sites (T). Tetracycline bone markers (orange and yellow) have been incorporated into the alveolar bone to demonstrate the bone turnover patterns. Note that the tension side is largely osteogenic, while the compression side is marked by cratering of both the bone and root, indicating resorption; however, some of the craters are also osteogenic, indicating that remodeling is taking place.

    Periodontal Ligament for Tooth Movement
The examples of tooth ankylosis and implants serve to demonstrate the essential role of the periodontal ligament in tooth movement. Ankylosed teeth have focal lesions characterized by bony bridges that eliminate the periodontal ligament in these areas. Similarly, implants with or without osseointegration also lack a periodontal ligament. In both instances, teeth are unresponsive to orthodontic tooth movement and dental drift. An appreciation of this has provided the impetus for the recent wide acceptance by orthodontists of mini-implants as temporary anchorage devices. It also explains why ankylosed deciduous teeth appear to submerge as adjacent teeth continue to adjust to vertical facial growth.

The specific underlying role of the periodontal ligament in tooth movement is not well-understood, but its unique biomechanical, cellular, and molecular natures are undoubtedly important. From a biomaterials perspective, the periodontal ligament is a complex, fiber-reinforced substance that responds to force in a viscoelastic and non-linear manner (Jonsdottir et al., 2006). This response is characterized by an instantaneous displacement, followed by a more gradual (creep) displacement that reaches a maximum after 5 hrs (van Driel et al., 2000), suggesting that fluid compartments within the periodontal ligament may play an important role in the transmission and damping of forces acting on teeth. The strains that are created in the periodontal ligament by force application clearly have biological consequences for the tissue itself, and possibly also for the other tooth-supporting tissues (i.e., alveolar bone and cementum).

Periodontal ligament cells respond to force by increases in cell proliferation and apoptosis. The relative extent to which these two competing processes occur controls the various cell populations in the periodontal ligament and reflects the specific biomechanics (Mabuchi et al., 2002).

The major fibrous components of the periodontal ligament extracellular matrix (collagen, tropoelastin, and fibronectin) also show enhanced expression following force application (Howard et al., 1998; Redlich et al., 2004a). Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a coordinated fashion to regulate collagen remodeling. The periodontal ligament expression levels of MMP-2, 8, 9, 13, and TIMPs 1-3 increase transiently during orthodontic tooth movement. However, these genes have different patterns of expression at compression and tension sites, suggesting that collagen remodeling is regulated differentially based on mechanics (Howard et al., 1998; Takahashi et al., 2003, 2006). This conclusion is given further support by the observations that tension prevents degradation of the matrix by inhibiting MMP-1 (Arnoczky et al., 2004), while relaxation of tension enhances extracellular matrix resorption (Von den Hoff, 2003). Enhanced expression of MMP-1 in periodontal ligament fibroblasts also may be the result of a direct effect of force on the gene (Redlich et al., 2004b).

Matrix proteoglycans are also altered in the periodontal ligament during orthodontic tooth movement. Periodontal ligament chondroitin sulfate (CS) and heparin sulfate (HS) increase during tooth movement and decrease in hypofunction. However, the complex patterns of CS and HS changes in tooth movement make interpretation of their roles difficult (Esashika et al., 2003). Hyaluronic acid (HA), present in the periodontal ligament, may bind with increased amounts of versican, a large HA-binding proteoglycan, and link protein localized at compressive sites, to create large hydrated aggregates. These may act either to limit tissue damage by dissipating excessive compressive forces, or to provide space to facilitate the migration of resorptive cells into these sites (Sato et al., 2002).

The Turnover of Adjacent Alveolar Bone
Frost’s pioneering descriptions of how bone turns over have provided researchers and clinicians with important concepts that have improved our understanding of numerous bony processes that were previously viewed as unrelated, including osteoporosis, fracture healing, mechanical usage, metabolic and genetic bone disease, and skeletal growth and development (Frost, 2001). These concepts can also provide important insights into the differences and similarities between dental eruption and orthodontics.

Bones turn over by two related, but distinct, processes Frost called ’modeling’ and ’remodeling’. Modeling is characterized by either osteogenesis or resorption that is sustained over a specific period of time and at precise bony surfaces. It results in skeletal shape change and translocation of hard-tissue structures. Modeling processes are prevalent in skeletal development, where individual bones move in relation to each other and change shape. The intra-osseous phase of tooth eruption can be considered to be primarily a process of alveolar bone modeling.

    Bone Resorption (Modeling) in Eruption
Given that the unerupted tooth is encased in alveolar bone, bone resorption is required for the tooth to erupt. In turn, osteoclast formation (osteoclastogenesis) is needed for an adequate number of osteoclasts to be present to resorb the alveolar bone.

A unique feature of bone resorption in the formation of an eruption pathway is that it can be uncoupled from tooth eruption—i.e., the tooth does not have to move for the eruption pathway to form. This observation lends support to the idea that the bone modeling in tooth eruption is genetically controlled, and not mechanically regulated by the eruption of the tooth. Immobilizing the erupting permanent third premolars in the mandible in the dog does not stop the formation of the eruption pathway (Cahill, 1969a). The osteoclasts resorbing the alveolar bone appear to arise from an influx of mononuclear cells (osteoclast precursors) into the dental follicle at a specific time prior to eruption, as shown in the dog (Marks et al., 1983), rat (Wise and Fan, 1989), and mouse (Volejnikova et al., 1997). In turn, osteoclast numbers increase on the alveolar bone surface at the same time as a result of fusion of these mononuclear cells to form osteoclasts (Marks et al., 1983; Wise et al., 1985).

At the ultrastructural level, the architecture of the alveolar bone reveals that bone resorption is occurring in the coronal region of the bony crypt prior to and during the intra-osseous phase of eruption. Specifically, in the dog, the architecture of the bone in the coronal region of the crypt appears scalloped (Marks and Cahill, 1986), a finding confirmed for the socket of the first mandibular molar of the rat (Wise et al., 2007).

Numerous experiments have confirmed that bone resorption is required for tooth eruption. Injection into rats of a bisphosphonate, pamidronate, which slows resorption, results in a delay in the time of molar eruption (Grier and Wise, 1998). Bafilomycin A₂, another agent that inhibits osteoclast activity, has also been reported to inhibit tooth eruption (Sundquist and Marks, 1994), although some of the toxic effects of this molecule may also affect eruption. Conversely, injecting colony-stimulating factor-1, a molecule that promotes osteoclastogenesis, accelerates the time of eruption (Cielinski et al., 1994, 1995).

Inhibition of the molecules that promote osteoclastogenesis can inhibit eruption. In knock-out mice devoid of receptor activator of nuclear factor kappa B (RANKL), the teeth do not erupt (Kong et al., 1999). In osteopetrotic rodents in which osteoclasts are either absent or non-functional, teeth do not erupt. Molecular analyses have shown that osteopetrotic mice (op/op) do not have functional colony-stimulating factor-1 (Felix et al., 1990; Wiktor-Jedrzejczak et al., 1990; Yoshida et al., 1990), and the osteopetrotic toothless (tl) rat is the result of a loss-of-function frameshift mutation in the CSF-1 gene (Dobbins et al., 2002; Van Wesenbeeck et al., 2002). Injection of CSF-1 into these animals at an early age prior to the onset of eruption will induce eruption (Iizuka et al., 1992).

    Bone Remodeling
Bone remodeling is a cyclic process that is a response to the need for continuous repair and renewal of the skeleton throughout life. Frost describes a basic multicellular unit that performs a coordinated series of events comprising the remodeling cycle. A remodeling cycle has four phases: activation, resorption, reversal, and formation. Although this sequence of events has been confirmed in numerous contexts and is widely accepted as the way that the skeleton repairs itself, the precise mechanisms controlling the basic multicellular units are not well-understood. The timing of the histological events occurring at compression sites in orthodontic tooth movement is consistent with a remodeling cycle (King et al., 1991b) (Fig. 2B). This, along with the abundant evidence for tissue damage at these cites, strongly suggests that remodeling is a prevalent bone turnover process at orthodontic compression sites.

One important consideration is how remodeling cycles are initiated. Much experimental evidence has linked bone remodeling to microdamage, and to subsequent increased cellular activity. Microcracks in bone caused by fatigue or trauma may play an important role in the initiation of remodeling cycles (Galleyv et al., 2006), because crack displacements are capable of tearing osteocyte cell processes, which may directly secrete bioactive molecules into the extracellular matrix, triggering a response (Hazenberg et al., 2006). The increased prevalence of microcracks at compression sites in orthodontic tooth movement further suggests that they are important in initiating orthodontic bone remodeling (Verna et al., 2004).

Another important bone remodeling concept is coupling between resorption and formation. Coupling mechanisms have been postulated as a means by which bone is neither lost nor gained during repair. The exact mechanism by which coupling is achieved is not well-understood, but is thought to be controlled by the release of paracrine molecules by the cells of the basic multicellular unit. During the early stages of repair in tooth movement, the occurrence of several paracrine factors (e.g., IGF-II, IGFBP-5 or -6) within lacunae and in cementoblasts suggests that these may be involved in controlling this remodeling sequence (Hazenberg et al., 2006).

Another related coupling issue involves the relative rates of resorption vs. formation. The former is quite rapid, while the latter is significantly slower. This has important consequences for bones that are undergoing extensive remodeling—for example, during the perimenopausal period (Recker et al., 2004). In these instances, bone formation cannot keep pace with the large amounts of resorption that are occurring, with the end result being net bone loss. The failure of formation to keep up with resorption during the extensive amount of remodeling at compression sites during orthodontic treatment also may explain the common clinical finding of tooth mobility and widening of the periodontal ligament space.

Alveolar bone resorption and formation appear not to be coupled in eruption—i.e., resorption occurs in the coronal portion of the alveolar bony crypt (socket), whereas bone formation occurs at the base of the socket. Moreover, if one process, such as bone formation, is blocked by temporarily impacting the erupting tooth, bone resorption continues, and an eruption pathway is formed (Cahill, 1969a). However, given that bone formation occurs rapidly at the base of the socket once the restraint on the erupting tooth is removed (Cahill, 1969b), one cannot fully eliminate the possibility that communication between the basal and coronal halves of the bony socket may occur and, in turn, perhaps influence the rate of bone formation or resorption occurring in the basal and coronal halves, respectively.

	MOLECULAR REGULATION OF OSTEOCLASTOGENESIS

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Background
Osteoclast biology underwent a revolution in the late 1990s, not only with the discovery of a critical set of molecules that regulated osteoclastogenesis, but also with the elucidation of how they interacted. In particular, a member of the TNF ligand family, RANKL, was initially found to be a membrane-bound protein present on osteoblasts and stromal cells, as well as on other cell types (Anderson et al., 1997; Wong et al., 1997; Yasuda et al., 1998a). Cell-to-cell signaling between cells with RANKL on their surfaces and osteoclast precursors carrying the receptor RANK induced both osteoclast formation and activation (Yasuda et al., 1999). Three isoforms of RANKL have since been identified, two of which are transmembrane proteins, whereas the third—RANKL3—is a soluble form (Ikeda et al., 2001).

The receptor for RANKL on osteoclast precursors is the receptor activator of NF-B (RANK), first identified by Anderson et al.(1997). In turn, CSF-1 is required to up-regulate RANK gene expression in osteoclast precursors (Arai et al., 1999), and this is one of the reasons that CSF-1 is required for osteoclastogenesis. The growth and differentiation of mononuclear pre-osteoclasts are also dependent upon CSF-1 (Stanley et al., 1983; Tanaka et al., 1993). Moreover, CSF-1 appears to have chemotactic properties for recruiting osteoclast progenitors (Wang et al., 1988; Bober et al., 1995; Que and Wise, 1997).

The cell-to-cell signaling involving the binding of RANKL to RANK results in recruitment of various members of TNF receptor-associated factors (TRAFs) within the osteoclast precursor, of which TRAF6 appears to be a key player (Darnay et al., 1999; Wong et al., 1999). For example, TRAF6 activates the signaling pathways for NFB and c-Fos (Boyle et al., 2003). Null mice devoid of the c-Fos gene have no osteoclasts, but they do have osteoclast precursors (Grigoriadis et al., 1994), and the same is true for mice devoid of the NFB genes (Franzoso et al., 1997; Iotsova et al., 1997). Moreover, in these null mice, the teeth do not erupt. Of particular interest regarding c-Fos is that RANKL signaling through c-Fos induces the interferon-β (IFN-β) gene in osteoclast progenitor cells, and the IFN-β synthesized negatively feeds back on the cells to inhibit the expression of c-Fos (Takayanagi et al., 2002).

TRAF6 also binds Src tyrosine kinase, which likely is the effector molecule in osteoclast activation, because it is required for cytoskeletal protein re-arrangement to form a ruffled border (Boyce et al., 1992). Src also appears to promote osteoclast survival by preventing apoptosis (Wong et al., 1999; Xing et al., 2001).

A means of either fine-tuning or inhibiting the stimulation of osteoclastogenesis is obviously needed. The molecule that does this is osteoprotegerin, a secreted glycoprotein that is a decoy-receptor for RANKL (Simonet et al., 1997; Tsuda et al., 1997; Yasuda et al., 1998b). Binding of osteoprotegerin to RANKL inhibits the cell-to-cell signaling that occurs between cells with RANKL on their membrane and osteoclast precursors, resulting in the inhibition of osteoclastogenesis (Yasuda et al., 1998b, 1999). In vivo, over-expression of osteoprotegerin in transgenic mice results in osteopetrosis and few osteoclasts, although TRAP-positive mononuclear cells (pre-osteoclasts) are present (Simonet et al., 1997). Injection of recombinant osteoprotegerin into mice also produces the same results (Simonet et al., 1997).

Fusion of the osteoclast precursors to form osteoclasts appears to require a transmembrane receptor molecule, dendritic cell-specific transmembrane protein (DC-STAMP) (Kukita et al., 2004; Yagi et al., 2005). Gene expression of DC-STAMP is induced in osteoclast precursors by RANKL, and inhibition of this expression by small interfering RNAs inhibits osteoclast formation (Kukita et al., 2004). DC-STAMP knockout mice also have no multi-nucleated osteoclasts, but do have mononuclear cells that are tartrate-resistant acid-phosphatase (TRAP)-positive (Yagi et al., 2005).

Another molecule that may affect fusion is secreted frizzled-related protein-1 (SFRP-1), a molecule that inhibits osteoclastogenesis (Häusler et al., 2004). This molecule also is secreted by the dental follicle, and in vitro osteoclastogenic assays show that, although it prevents osteoclast formation, increasing concentrations of SFRP-1 result in increased numbers of TRAP-positive mononuclear cells (Liu and Wise, 2007). Thus, SFRP-1 may act to prevent osteoclastogenesis by preventing fusion of the precursor cells. Molecules involved in osteoclastogenesis can be found in Table 1.

Given that the dental follicle is required for eruption, and that osteoclast precursors are being recruited to it, what are the molecular events occurring in the follicle to regulate the bursts of osteoclastogenesis? Consider the rat mandibular first molar as the model: An early event is the recruitment of the mononuclear cells to the dental follicle. Gene expression and immunostaining studies show that CSF-1 is maximally expressed in the rat follicle at day 3 post-natally, followed by a precipitous drop in subsequent days (Wise et al., 1995). Monocyte chemotactic protein-1 (MCP-1) also shows a similar expression pattern (Que and Wise, 1997). Both CSF-1 and MCP-1 are chemokines for monocytes (Rollins et al., 1988; Wang et al., 1988; Yu and Graves, 1995), and, in subsequent in vitro studies, we demonstrated that both CSF-1 and MCP-1 are secreted by the dental follicle cells and are chemotactic for monocytes (Que and Wise, 1997). Gene microarray studies have confirmed this enhanced expression of CSF-1 and MCP-1 and have detected an increased expression of one other chemokine, endothelial monocyte–activating polypeptide 2 (EMAP-II), in the follicle at days 3 and 9 (Liu and Wise, 2007). Current studies are under way to determine if EMAP-II is also secreted by the follicle cells and is chemotactic for mononuclear cells. It should also be noted that once osteoclast precursors reach the dental follicle, the precursors themselves might elicit paracrine chemotactic signals, such as the chemokine CCL9 (e.g., see Yang et al., 2006).

The correlation between maximal expression of these chemokines in the rat dental follicle and the maximal number of mononuclear cells at day 3 is striking. Although correlation is not always causal, such a correlation in another species strongly suggests that these chemokines are central to the recruitment of mononuclear pre-osteoclasts to the follicle. In the mouse, the time of maximal mononuclear cell numbers in the follicle is at day 5, and that is the time that both CSF-1 and MCP-1 genes are maximally expressed in the follicle of the mouse (Wise et al., 1999).

Enhancement of CSF and MCP-1 gene expression in the follicle at this early time post-natally, the day 3 rat and the day 5 mouse, may arise from the expression of other molecules in the stellate reticulum, the epithelium adjacent to the dental follicle. For example, molecules such as transforming growth factor-beta 1 (TGF-β₁) and interleukin-1 (IL-1) are expressed maximally early in the rat dental follicle (Wise et al., 2002). In turn, TGF-β₁ and IL-1 enhance MCP-1 expression in the dental follicle (Que and Wise, 1998), enhance synthesis and secretion of MCP-1 by the follicle cells (Wise et al., 1999), and, in the case of IL-1, enhance the chemotactic ability of the dental follicle cells (Wise et al., 1999). IL-1 enhances the transcription of the CSF-1 gene in the dental follicle cells in vitro (Wise and Lin, 1994), and injection of IL-1 enhances CSF-1 expression in vivo in the follicle (Wise, 1998).

It is of interest to note that, almost 20 years ago, Cahill et al.(1988) proposed that a "clock" existed in the enamel organ that regulated the cellular events of eruption. The stellate reticulum is a major component of the enamel organ adjacent to the dental follicle, and one could speculate that it is the secretion of such molecules as IL-1 and/or TGF-β₁ with their subsequent effect on the dental follicle that initiates eruption. However, in null mice devoid of the IL-1 receptor gene, the teeth do erupt, although there is a slight delay in eruption (Huang and Wise, 2000). Thus, eruption can occur without the IL-1 signal.

Another molecule localized to the stellate reticulum is parathyroid-hormone-related protein (PTHrP), and no tooth eruption occurs in the absence of its expression in the stellate reticulum (Philbrick et al., 1998). However, we have recently shown that PTHrP is maximally expressed in the stellate reticulum at a later date (days 7–9), and thus may not initiate the onset of eruption (Yao et al., 2007).

Continuing with the major burst of osteoclastogenesis, after the osteoclast precursors have been recruited to the dental follicle, how does the dental follicle initiate and regulate osteoclast formation? The first clue came from studies showing that the osteoprotegerin gene was constitutively expressed in the dental follicle of either the rat or mouse (Wise et al., 2000). Most striking, however, was the fact that the osteoprotegerin was down-regulated in the dental follicle at day 3 in the rat and at day 5 in the mouse (Wise et al., 2000). In vitro, either CSF-1 or parathyroid-related protein (PTHrP) could decrease osteoprotegerin gene expression in the follicle cells in both a time-dependent and concentration-dependent fashion (Wise et al., 2000).

The decrease in osteoprotegerin expression in the rat dental follicle would allow maximal osteoclastogenesis to occur at day 3, as is indeed seen. The maximal expression and secretion of CSF-1 at day 3 are likely the reason for the down-regulation of osteoprotegerin at day 3. To determine if CSF down-regulated osteoprotegerin in vivo, we compared osteopetrotic toothless (tl) rats, deficient in CSF-1, with their normal littermates at given ages (days 1, 3, 5, 7, 9, 11), to determine if the osteoprotegerin expression was greater in the tl rat. If CSF-1 does inhibit osteoprotegerin gene expression, the absence of functional CSF-1 would result in a greater expression of osteoprotegerin in the tl rats than in the normal littermates. This indeed was the finding, and demonstrated that CSF-1 down-regulated CSF-1 in vivo (Wise et al., 2005). In conjunction with this, in vitro studies also showed that transfecting the dental follicle cells with a short interfering RNA specific for CSF-1 mRNA resulted in an up-regulation of osteoprotegerin expression (Wise et al., 2005).

Although the drop in osteoprotegerin in the dental follicle is viewed as the key regulatory event allowing osteoclastogenesis to occur at day 3, some RANKL, as well as the CSF-1 present, is needed to drive osteoclast formation. Laser capture microdissection and RT-PCR studies showed that RANKL was expressed in the dental follicle in vivo (Yao et al., 2004), and a subsequent real-time RT-PCR study showed that RANKL was expressed in the dental follicle post-natally, with maximum expression at day 9 (Liu et al., 2005). The fact that RANKL is present at day 3, but not maximally expressed until later, reinforces the concept that the decrease in osteoprotegerin expression effected by CSF-1 at day 3 is critical for enabling the major burst of osteoclastogenesis to occur; i.e., only by decreasing osteoprotegerin would a favorable ratio of RANKL/osteoprotegerin for osteoclastogenesis be established.

Recent gene microarray studies suggest that another inhibitor of osteoclastogenesis, SFRP-1, is present in the dental follicle and is down-regulated at days 3 and 9 post-natally (Liu and Wise, 2007). In vitro osteoclastogenesis assays show that maximal inhibition of osteoclast formation occurs when both anti-osteoprotegerin and anti-SFRP are present, suggesting that osteoprotegerin and SFRP may use different mechanisms to inhibit osteoclastogenesis (Liu and Wise, 2007). Regardless, the fact that both osteoprotegerin and SFRP gene expression are down-regulated at day 3 again emphasizes that the reduction of osteoclast inhibitors is critical for the major burst of osteoclastogenesis to occur.

In summary, Fig. 3 qualitatively depicts the levels of gene expression, in the dental follicle, that initiates and regulates the major burst of osteoclastogenesis at day 3 for the first mandibular molar of the rat. Maximal levels of MCP-1 and CSF-1 at day 3 promote the recruitment of osteoclast precursors to the dental follicle, where CSF-1 and RANKL can then stimulate osteoclastogenesis. Most importantly, the high level of CSF-1 reduces osteoprotegerin expression, such that the inhibition to osteoclastogenesis is reduced. The level of another inhibitor, SFRP1, is also reduced, but it is not yet known what molecule inhibits SFRP1.

View larger version (17K): [in this window] [in a new window]   Figure 3. Graph depicting the expression of genes in the dental follicle of the rat 1st mandibular molar at various days post-natally. At day 3, the major burst of osteoclastogenesis, osteoprotegerin is down-regulated such that a favorable RANK/osteoprotegerin ratio is established to promote osteoclastogenesis. At this time, CSF-1 is maximally expressed, both to down-regulate osteoprotegerin expression and to promote osteoclast precursor recruitment and osteoclastogenesis. At day 10, the minor burst of osteoclastogenesis, VEGF is maximally expressed to stimulate RANK expression on osteoclast precursors, as well as to interact with RANKL and CSF-1 to promote osteoclastogenesis. At this time, RANKL is up-regulated such that a favorable RANKL/osteoprotegerin ratio could exist to stimulate this minor burst of osteoclastogenesis.

In the major burst of osteoclastogenesis, CSF-1 appears to play a role in recruiting osteoclast precursors, depressing osteoprotegerin gene expression in the dental follicle, stimulating proliferation of osteoclast precursors, and stimulating RANK production in the osteoclast precursors. VEGF cannot do all of this. It has been reported that it can recruit osteoclasts to the site of injection of VEGF in osteopetrotic mice (Niida et al., 1999; Kaku et al., 2000). Whether it can recruit osteoclast precursors to the dental follicle is unknown. It can up-regulate the expression of RANK in endothelial cells (Min et al., 2003), and we recently have shown that it can up-regulate RANK expression in osteoclast precursors (Yao et al., 2006). This is likely one of its major roles for the minor burst of osteoclastogenesis.

VEGF appears not to down-regulate osteoprotegerin gene expression, because the constitutive level of osteoprotegerin expression during the minor burst of osteoclastogenesis does not decrease (see Fig. 3). In vitro, osteoclastogenesis assays also show that, in the presence of RANKL, purified spleen mononuclear cells are not induced to form osteoclasts to any significant degree when VEGF is added (Yao et al., 2006). In contrast, osteoclast formation is induced if CSF-1 is added to RANKL, and even greater numbers are seen if both CSF-1 and VEGF are present (Yao et al., 2006). Finally, VEGF cannot stimulate proliferation of the osteoclast precursors in vitro (Yao et al., 2006).

In essence, VEGF likely substitutes fully for CSF-1 to induce RANK formation. The small amount of CSF-1 present at day 10 likely is enough to interact with VEGF to help promote the minor burst of osteoclastogenesis.

The RANKL gene expression in the dental follicle is maximally up-regulated on days 9–11 (Liu et al., 2005). Although it is not known what molecule(s) may up-regulate RANKL expression in the dental follicle at this time, TNF- is a possible candidate, because it does up-regulate RANKL gene expression in the dental follicle cells in vitro (Liu et al., 2005), and because it is also maximally expressed at day 9 (Wise and Yao, 2003b), a time that correlates with the maximal RANKL expression.

That RANKL production in the dental follicle affects alveolar bone resorption is supported by studies in which mice null for RANKL were transfected with a CD4 enhancer to drive expression of RANKL in B- and T-lymphocytes, but not in the dental follicle cells. In such rescued mice, bone resorption was seen in the long bones, but no alveolar bone resorption or tooth eruption was seen (Odgren et al., 2003). Thus, it is the production of RANKL in the dental follicle itself that appears to be needed for the promotion of osteoclastogenesis and resorption of alveolar bone.

It is likely that the maximal expression of RANKL at days 9–11 creates a favorable ratio for the minor burst of osteoclastogenesis, despite the high levels of osteoprotegerin present (Fig. 3). With the VEGF and a small amount of CSF-1 also present, in vitro studies show that osteoclastogenesis can occur (Yao et al., 2006).

Although not shown in Fig. 3, another putative eruption molecule, PTHrP, may play some role in the minor burst of osteoclastogenesis. Laser capture microdissection and RT-PCR studies have shown that PTHrP is maximally expressed in the stellate reticulum at days 7–9, and in vitro PTHrP can enhance VEGF gene expression in the dental follicle cells (Yao et al., 2007). Others have also suggested that PTHrP can up-regulate RANKL expression in dental follicle cells (Nakchbandi et al., 2000), but we have not been able to show this in our dental follicle cell lines. Regardless, PTHrP may have other functions in tooth eruption as well. For example, in PTHrP-gene knockout mice or in tooth germs treated with an antisense oligonucleotide against PTHrP, there are few osteoclasts around the tooth germ, and bone spicules invade the tooth germ (Liu et al., 2000; Kitahara et al., 2002). Thus, the authors suggest that PTHrP may protect the tooth germs from bone invasion and subsequent ankylosis. PTHrP may also affect bone formation (osteogenesis), as will be discussed later.

Although this review has focused on the chronology of the expression of tooth eruption genes in the regulation of osteoclastogenesis, the regional localization of the genes within the dental follicle may be of equal importance. One only has to examine the ultrastructure of the alveolar bony crypt in which the unerupted tooth resides to see that two very different activities are occurring at opposite poles of the crypt—i.e., bone resorption in the coronal one-half and bone formation in the basal (apical) one-half (Fig. 4). Bone architecture reflects the physiological state of the bone (Boyde and Hobdell, 1969), and scanning electron microscope studies of the bony crypt of the 3rd and 4th premolars of the dog showed that the coronal region of the bony crypt appeared scalloped (indicating bone resorption), whereas the basal region was trabecular (indicating bone formation) (Marks and Cahill, 1986). Similar findings were observed for the alveolar bony crypt of the first mandibular molar of the rat (Wise et al., 2007). Thus, it was postulated that the coronal one-half of the dental follicle would regulate bone resorption, and the basal one-half of the dental follicle would regulate bone formation, a hypothesis supported by the finding that removal of only the coronal one-half of the follicle would prevent bone resorption and tooth eruption (Marks and Cahill, 1987).

View larger version (121K): [in this window] [in a new window]   Figure 4. High-power SEM view of a portion of the wall of the alveolar bony crypt of a 1st mandibular rat molar at day 3 post-natally, extending from the coronal region (C) of the wall to the basal region (B). Although only a small area of the large coronal region is shown, note elongated depressions (D) that give a scalloped appearance to the bone in that region, whereas in the basal region the bone consists of many narrow trabeculae (T). Scalloped bone is undergoing resorption, whereas trabecular bone reflects bone formation. Between the two is an intermediate region (I) of bone that is relatively smooth. Such smooth bone is more inert, or neutral, reflecting neither growth nor resorption.

Finally, in view of the fact that the dental follicle differentiates into the periodontal ligament, are any of the eruption genes of the dental follicle that regulate osteoclastogenesis expressed in the periodontal ligament? Numerous reports have indicated that key osteoclastogenic molecules—such as RANKL (Kanzaki et al., 2001; Hasegawa et al., 2002; Fukushima et al., 2003), osteoprotegerin (Sakata et al., 1999; Kanzaki et al., 2001; Wada et al., 2001; Hasegawa et al., 2002), and VEGF (Oyama et al., 2000)—are expressed in the periodontal ligament. The osteoprotegerin is secreted in vitro by the periodontal ligament fibroblasts and can inhibit osteoclast formation (Wada et al., 2001). In essence, it appears that the constitutive synthesis of osteoprotegerin by the periodontal ligament would serve to prevent osteoclastogenesis of the alveolar bone, such that the periodontal ligament attachment would remain intact. Only during a period of tooth eruption would the osteoprotegerin expression need to be inhibited such that bone resorption could occur. In disease states such as periodontitis, however, RANKL levels are up-regulated and osteoprotegerin levels down-regulated (Crotti et al., 2003; Liu et al., 2003; Mogi et al., 2004), such that the alveolar bone is resorbed. In essence, periodontitis mimics, to some extent, the osteoclastogenic events of tooth eruption.

In Orthodontic Tooth Movement
Osteoclastogenesis in orthodontic tooth movement is initiated by two related changes brought about by the application of force: tissue damage, with the subsequent production of inflammatory processes in the periodontal ligament; and deformation of the alveolar process. Osteoclasts and committed osteoclast progenitor cells, identified by the synthesis of tartrate-resistant ATPase and H(+)-ATPase immunohistochemistry, appear at sites of compression within days after forces are applied. Osteoclast induction, represented by mononuclear pre-osteoclasts, first occurs in vascular and marrow spaces of the alveolar crest, followed by increases in the periodontal ligament space (Yokoya et al., 1997; Rody et al., 2001). Their numbers correlate with finite element method (FEM) predictions of strains in the periodontal ligament and alveolar bone, with compression sites showing more than tension sites (Kawarizadeh et al., 2004). Increases in pro-inflammatory cytokines (IL-1, 6, 8, and TNF) also correlate well with this distribution (Alhashimi et al., 2001; Bletsa et al., 2006; Lee et al., 2007), suggesting that cytokines are important initiators of osteoclastogenesis in tooth movement. Experiments have also demonstrated that these cytokines interact synergistically with bradykinin and thrombin in prostaglandin biosynthesis, thereby mediating inflammatory bone resorption (Marklund et al., 1994; Ransjo et al., 1998). There is also evidence that local administration of rhVEGF markedly enhances the number of osteoclasts at pressure sites during orthodontic tooth movement in osteopetrotic (op/op) mice (Kaku et al., 2001), and that treatment with anti-VEGF antibody reduces osteoclast numbers and the amount of tooth movement (Kohno et al., 2005). Analysis of these data suggests that the VEGF-CSF-1 mechanism, previously described in osteoclastogenesis associated with tooth eruption, may also be important in orthodontic tooth movement.

Changes in RANK, RANKL and osteoprotegerin have been demonstrated in the tooth-supporting tissues during orthodontic tooth movement (Oshiro et al., 2002), with evidence of RANKL stimulation and osteoprotegerin inhibition of osteoclastogenesis (Kanzaki et al., 2001). Compressive force up-regulates RANKL through a PGE₂ pathway, supporting osteoclastogenesis (Kanzaki et al., 2002), while local osteoprotegerin gene transfer to the tooth-supporting tissues inhibits RANKL-mediated osteoclastogenesis and tooth movement (Kanzaki et al., 2004). Increases in RANKL and the decreases in osteoprotegerin have also been demonstrated in cases of severe orthodontic root resorption, suggesting that this mechanism may be important in this negative sequelum of orthodontic treatment (Yamaguchi et al., 2006).

Clearance of osteoclasts from compression sites occurs between 5 and 7 days following appliance activation in the rat (King et al., 1991b). This is initiated in part by osteoclast apoptosis, followed by secondary necrosis (Noxon et al., 2001). Physical forces act through specific receptor-like molecules—such as integrins, focal adhesion proteins, and the cytoskeleton—to activate certain protein kinase pathways (p38 MAPK and JNK/SAPK), which in turn amplify the signal and activate caspases, promoting osteoclast apoptosis. The cell phenotype and the character of the physical stimuli determine which pathways are activated and, consequently, allow for variability in response to a specific stimulus in different cell types (Hsieh and Nguyen, 2005). In addition to osteoclasts, osteocytes have been shown to undergo apoptosis at orthodontic compression sites (Hamaya et al., 2002), but the details of how these two mechanisms may differ remain unclear. The latter is related to disuse (Bakker et al., 2004), suggesting that the unloading of the principal fibers of the periodontal ligament at these sites may be important.

	MOLECULAR REGULATION OF OSTEOGENESIS

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In Tooth Eruption
As described earlier, the architecture of the alveolar bony crypt displays different regions of bone activity, as seen by SEM. In the sockets of the 3rd and 4th mandibular premolars of the dog, 3 distinct regions exist: (1) the coronal (superior) one-half, consisting of scalloped bone (resorption occurring); (2) the basal (apical) one-half, consisting of trabecular bone (formation occurring); and (3) a narrow smooth area between the two halves that is an inactive region (Marks and Cahill, 1986; Marks et al., 1994). A similar SEM morphology is seen for the socket of the first mandibular molar of the rat (Fig. 3), except that the smooth area of bone is not always as well-defined (Wise and Yao, 2006; Wise et al., 2007).

The manner in which the dental follicle might regulate this disparate bone activity at opposite poles of the bony crypt was first suggested by Marks and Cahill (1986), who postulated that the coronal region of the dental follicle might regulate alveolar bone resorption, whereas the basal region of the dental follicle would regulate bone formation (osteogenesis). They followed this up with a study in which they surgically removed either the coronal one-half or the basal one-half of the dental follicle of the dog premolar and examined the effect on eruption. Removal of the coronal one-half of the dental follicle resulted in no alveolar bone resorption and no tooth eruption, and removal of the basal one-half resulted in no bone growth and no tooth eruption (Marks and Cahill, 1987). These studies dramatically demonstrated that both bone resorption and bone formation are required for eruption. Equally important, it appeared that the coronal portion of the dental follicle regulated bone resorption, whereas the basal portion of the dental follicle regulated osteogenesis.

The molecular regulation of osteogenesis by the dental follicle has recently begun to be elucidated, thanks in large part to laser capture microdissection, which allows one to excise specific regions of the dental follicle of different ages and then examine gene expression of these excised regions using real-time RT-PCR. Thus, a molecule that promotes osteoblast formation and osteogenesis, BMP-2 (Wang et al., 1990; Chen et al., 1998; Gori et al., 1999) was examined. BMP-2 was expressed in the follicle (Wise et al., 2004), and comparison of the coronal vs. basal halves for a given age showed that, beginning at day 3 post-natally, BMP-2 was expressed more in the basal one-half than in the corresponding coronal one-half for a given day, other than for day 7 (Wise and Yao, 2006).

The correlation between BMP-2 expression in the dental follicle and bone growth at the base of the socket further supports the view that the basal one-half of the dental follicle regulates osteogenesis, and that BMP-2 is a critical molecule needed for osteogenesis. In a recent SEM study of the alveolar bony crypt of the rat 1st mandibular molar, trabecular bone (osteogenesis) was seen at the base of the crypt beginning at day 3, and extensive trabecular bone was seen at the base at day 9 (Wise et al., 2007). At both of these times, the level of BMP-2 gene expression in the basal half of the dental follicle exceeded that in its coronal counterpart (Wise and Yao, 2006). However, at day 7, the base of the crypt was relatively smooth, and it is on this day in which there was no significant difference between the coronal and basal halves in terms of BMP-2 gene expression (Wise and Yao, 2006; Wise et al., 2007). Thus, a strong correlation exists between BMP-2 expression in the basal one-half of the dental follicle and the presence of trabecular bone (osteogenesis) in the basal portion of the socket.

The presence and/or role of other potential osteo-inductive molecules in the dental follicle has not yet been examined. The expression of a critical transcription factor for osteoblast differentiation, core-binding factor a1 (Cbfa1) or Runx2, has been observed in the dental follicles of mice (D’Souza et al., 1999; Bronckers et al., 2001). Although mice null for Cbfa1 die at birth, heterozygotes, Cbaf1 (+/–), sometimes display a delay or failure of eruption (see review by Wise et al., 2002). Although Cbaf1 may be expressed in the dental follicle, the eruption delays in heterozygotes may be due to osteoblast defects. Regardless, the importance of osteogenesis in eruption is again emphasized.

Finally, the significance of osteogenesis in eruption, and an indirect molecular regulation of it, comes from studies of membrane-type 1 matrix metalloproteinase (MT1-MMP). Mice deficient in MT1-MMP display delayed tooth eruption (Beertsen et al., 2002; Bartlett et al., 2003). In both studies, alveolar bone resorption occurs, but alveolar bone growth does not. MT1-MMP degrades collagens I, II, and III, as well as other extracellular matrix molecules (d’Ortho et al., 1997), which, in turn, affects the remodeling of bone. In particular, Beertsen et al.(2002) found that periodontal ligament fibroblasts in the MT1-MMP-deficient mice show a large accumulation of phagosomes containing collagen fibrils. Thus, in the periodontal ligament (a dental follicle derivative), an appropriate remodeling of its connective tissue and the bone interface likely is needed for alveolar bone formation to occur (Beertsen et al., 2002).

In Orthodontic Tooth Movement
Tensile strains determine osteogenic activity, and the nature of the applied loads determines osteoblast recruitment (Fig. 2B). Static loads do not seem to play an important role in skeletal osteogenesis. Instead, osteogenesis is driven by bouts of loading above a threshold, and the most important characteristics of those loads are their strain rates, amplitudes, and durations (Forwood and Turner, 1995). At first, osteogenesis related to tooth movement seems unusual, because many orthodontic appliances are designed to deliver static, or slowly dissipating, loads. However, it is important to realize that the dentition is exposed to multiple changing loading bouts during mastication, swallowing, and speech, suggesting that the loads applied to the dentition are rarely static.

Much like tooth eruption, osteogenesis associated with orthodontics is mediated by various osteoinductive molecules. In general, most of these molecules are regulated by tensile strains and act by stimulating osteoblast progenitor cell proliferation in the periodontal ligament, subsequent bone formation, and the inhibition of bone resorption. Molecules that have been linked in this way to orthodontic tooth movement include TGFβ (Brady et al., 1998), various BMPs (Mitsui et al., 2006), bone sialoprotein (BSP) (Domon et al., 2001), and epidermal growth factor (EGF) (Guajardo et al., 2000; Gao et al., 2002). Although the precise mechanisms at work in orthodontic osteogenesis have not been extensively examined, reasonable inferences can be made from the extensive body of literature on bone mechanotransduction that will be discussed in the next section of this review.

	UNDERLYING BIOLOGICAL AND BIOMECHANICAL MECHANISMS

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Motive Force of Tooth Eruption
In discussions of tooth eruption, the use of the word "force" must be used carefully. Eruption is both a physiological and developmental event, and, as such, these events are the products of biological processes that may include growth, differential growth, apoptosis, cell migration, etc. In some instances, force may be a secondary event resulting from a biological process, but it is imperative that the underlying biological mechanisms be recognized as the required elements in eruption.

What are the biological mechanisms that result in the tooth emerging from the bony crypt in which it is encased, such that it ultimately reaches its occlusal plane? For the intra-osseous phase of eruption, in which the tooth moves out of its bony crypt to pierce the gingiva, the two processes discussed extensively in this review—osteoclastogenesis and osteogenesis—are required. Without bone resorption as a result of osteoclastogenesis, no eruption pathway forms, and the tooth cannot escape its bony crypt, as seen in osteopetrotic rodents or experimentally where alveolar bone resorption is inhibited (see review by Wise et al., 2002). Without alveolar bone formation, teeth do not erupt (Marks and Cahill, 1987; Beertsen et al., 2002; Bartlett et al., 2003).

Alveolar bone formation at the base of the tooth socket during tooth eruption has long been known to occur, as demonstrated elegantly in studies in which dog premolars were temporarily impacted (Cahill, 1969b). After release, there was extensive bone growth at the base of the socket as the teeth erupted. Later studies with microradiography and fluorescence microscopy also demonstrated alveolar bone growth at the base of the crypt (Pilipili et al., 1995).

A detailed SEM study of the alveolar bony crypt of the first mandibular molar of the rat confirmed that extensive bone growth occurs at the base of the crypt during the intra-osseous phase of eruption (Wise et al., 2007). Beginning at day 3, trabecular bone was seen at the base of the crypt, and by day 9 the crypt began to be reduced in depth as a result of this bone formation. By day 14, the bone almost filled the length of the crypt, to form the interradicular septum (Fig. 5). This extensive growth of the interradicular septum has also been observed in human molars (Sicher, 1942). Thus, in essence, this deposition of new bone only at the base of the crypt during the intra-osseous phase of eruption leaves no place for the tooth to go but coronally, toward the eruption pathway (Fig. 5).

View larger version (126K): [in this window] [in a new window]   Figure 5. Low-power SEM view of the alveolar bony crypt of the rat 1st mandibular molar at day 14, showing the extensive bone growth that has occurred at the base, especially in the region of the interradicular septum (IR). Four pits represent where the 4 roots reside—mesial (M), distal (D), mesiolabial (L), and mesiobuccal (B). A prominent interdental septum (ID) is present that separates the crypt of the first molar from the 2nd molar (2M). Note that all the newly formed bone, such as seen in the IR or ID, is trabecular.

1. root elongation as a force of eruption—Rootless teeth can erupt (Gowgiel, 1961; Marks and Cahill, 1984);
   
2. periodontal ligament as a force of eruption—In the rat molar, the dental follicle does not become organized into a periodontal ligament and attach to the cementum and alveolar bone until the intra-osseous phase of eruption is complete (Wise et al., 2007). The same is true for dog premolars (Cahill and Marks, 1982). In addition, an inert metal replica can erupt, and there is no periodontal ligament attachment to it (Marks and Cahill, 1984). Transection of fibers in the dental follicle prior to the onset of eruption does not affect eruption rates or movement (Cahill and Marks, 1980); and
   
3. vascular pressure—Regional changes in vascular pressure have long been proposed as a force of eruption, but the evidence for this is both inconclusive and contradictory (see review by Cahill et al., 1988; Marks and Schroeder, 1996). In more recent studies, Cheek et al.(2002) showed, in human 2nd premolars, that injection of a vasodilator above the root apex caused a transient increase (30 min) in the rate of eruption, whereas injection of a vasoconstrictor caused a decrease or intrusion. Similarly, in rat mandibular incisors, systemic infusion of angiotensin II increased mean arterial pressure, decreased regional blood flow, and decreased the eruption rate (Shimada et al., 2004). Conversely, a positive correlation was found between the eruption rate and increased regional blood flow.
   

Unfortunately, it is difficult to reconcile these studies with the fact that an inert object minus pulp and roots can erupt—i.e., there are no vessels in the pulp to affect eruption (Marks and Cahill, 198