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Periosteal growth at human mandibular ramus is characterized by bone apposition at the posterior border and resorption at the anterior border. Molecular control of this regional variation is unclear. This study examined the expression of several molecules involved in bone apposition/resorption at these regions in vivo and in vitro. By using growing pigs as a model, the periosteal growth was assessed at the mandibular ramus by vital staining and histological observations. In parallel, periosteal tissues were harvested and pulverized for RNA and protein extraction. Periosteal cells were also isolated, expanded in osteogenic media, and subjected to a single dose of dynamic tensile strain (0, 5, or 10% magnitude at 0.5 Hz) to examine their responses to mechanical loading. Real-time RT-PCR and Western blot analyses were used to examine mRNA and protein expression from periosteal tissues and cultured cells. Histological observation confirmed an anterior-resorption/posterior-apposition pattern in the pig mandibular ramus. Both in vivo tissue and in vitro cells demonstrated greater mRNA expression of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio and bone morphogenetic protein 2 (BMP2) at the anterior region, while OPG expression at the anterior region was lower than the posterior region. In response to the application of a single dose of dynamic tensile strain, cultured periosteal cells appeared to change the expression profile of osteogenic markers but not that of RANKL/OPG and BMP2. These findings suggest that the unique regional variation of periosteal activity at the mandibular ramus is regulated by a differential expression of RANKL/OPG ratio (likely through differential induction of OPG) and BMP2. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.
Human mandibular growth is mainly through endochondral bone formation at the condyles and intramembranous bone formation at the periosteum (Enlow and Hans,1996). While condylar growth provides the overall elongation and rotation of the mandible (Bjork and Skieller,1983), periosteal activity causes extensive surface modeling/remodeling or reshaping of the mandible (Enlow and Hans,1996). One striking feature of mandibular periosteal activity is at the ramus, where bone resorption takes place at the anterior (rostral) border whereas bone apposition at the posterior (caudal) border (Robinson and Sarnat,1955; Seiton and Engel,1969; Hans et al.,1995). At the cell level, osteogenic proliferation is concentrated at the appositional posterior ramal region, whereas osteoclasts are mainly present at the resorptive anterior region (Ochareon and Herring,2007). However, the molecular mechanisms related to these differential periosteal activities are mostly unknown.
Recruitment and maturation of osteoclasts for bone resorption require two important molecules, colony-stimulating factor (Felix et al.,1990; Cecchini et al.,1997) and receptor activator of NF-κB ligand (RANKL) (Hsu et al.,1999; Wada et al.,2006), both of which are expressed by osteoblasts. The binding of RANKL to the RANK receptor on osteoclasts activates osteoclast differentiation and bone resorption. On the other hand, osteoblasts also produce osteoprotegerin (OPG), a decoy receptor for RANKL, to inhibit RANK activation by RANKL. The ratio of RANKL to OPG expression determines the extent of bone resorption activity. Accordingly, the differential periosteal activity between the anterior and posterior ramal borders may result from the variations in the relative RANKL/OPG expression.
Clarifying the molecular mechanisms related to the regional variation of the mandibular periosteal activity is important for a better understanding of mandibular growth, as well as for using periosteal cells in tissue engineering applications. Mandibular periosteal cells have been recently used to augment the maxillary sinus floor before dental implant placement (Schmelzeisen et al.,2003) and to enhance bone repair at calvarial defects (Miyamoto et al.,2004; Sakata et al.,2006). In these studies, periosteal cells were collected either from the lateral ramal surface (Schmelzeisen et al.,2003) or from the anterior ramal surface (Miyamoto et al.,2004; Sakata et al.,2006). Access to the lateral ramal surface requires a surgical procedure more invasive than access to the anterior ramal surface, which can be easily obtained during a routine wisdom tooth extraction. Despite easy access, given that the anterior periosteal surface is dominated by bone resorption (Enlow and Hans,1996), the anterior mandibular ramal surface may not provide an optimal periosteal cell source for bone engineering unless their molecular bone resorptive profile is reversed by in vitro treatments.
One treatment that can potentially change the phenotype of ramal periosteal cells in vitro is to apply mechanical stretching (tensile strain). During normal mandibular growth, both the fibrous (Grimm and Katele,1979) and the osteogenic (Ochareon and Herring,2007) layers of the periosteum are displaced toward the appositional posterior border. Such displacement is thought to be caused by mechanical stretching generated during bone elongation (Frankenhuis-van den Heuvel et al.,1992). In addition to physical displacement, mechanical stretching may also change the phenotype of periosteal cells, as abundant evidence has shown that mechanical stretching activates osteoblasts in a number of signaling pathways (Wang et al.,2001; Armstrong et al.,2007; Sunters et al.,2010) and promotes osteogenic differentiation of cultured mesenchymal stem cells (Ward et al.,2007; Huang et al.,2009). In this study, we sought to characterize the molecular basis of mandibular ramal periosteum in relation to the juxtaposing bone apposition/resorption activities and examine the effect of mechanical stretching on ramal periosteal cells during in vitro expansion.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
Similar to humans, the pig mandibular ramus grows in an anterior-resorption and posterior-apposition pattern (Fig. 1A–F), confirming that the pig is an acceptable model to study the unique ramal periosteal growth in the human (Robinson and Sarnat,1955; Ochareon and Herring,2007). Therefore, the findings obtained from this study about pig molecular changes at the ramal periosteum provide a new understanding of periosteal growth in the human mandible, an area important for clinical orthodontics and craniofacial orthopedics.
In this study, the expression of several molecules involved in bone resorption/apposition regulation was quantified from harvested periosteal tissues and cultured periosteal cells. When compared with direct tissue processing, in vitro expansion and passaging, especially with the use of serum and supplements for osteogenic induction, can potentially impact gene expression (Tamama et al.,2010). Relatively, large intersample variability was observed in the expression of some molecules (Fig. 3). The samples used in this study were cells from different passages (passage three to five), rather than triplicates from the same passage. Very likely, this variability is at least in part contributed by changes caused by passaging. On the other hand, despite such intersample variability, we found that periosteal tissues and cultured cells consistently showed significantly greater expression of BMP2 and RANKL/OPG ratio at the anterior region than at the posterior region (Figs. 2, 3), suggesting that these genes may be involved in regulating differential periosteal activities at these regions.
Increased RANKL/OPG ratio accompanies bone resorption involved in a variety of processes such as pathological osteolysis (Grimaud et al.,2003; Tay et al.,2004), periodontitis (Crotti et al.,2003), tooth eruption (Heinrich et al.,2005), and orthodontic tooth movement (Yamaguchi,2009). The current data adds physiologic mandibular periosteal growth to that list. During normal growth, infection or inflammation is absent at the anterior ramal surface. Therefore, an increased RANKL/OPG ratio is not caused by infection or inflammation. Periosteal tissue from the posterior region had significantly greater expression of OPG than that from the anterior region, while their expression of RANKL was similar (Fig. 2). Cultured posterior periosteal cells without mechanical stretching also demonstrated a tendency of greater OPG expression than anterior cells, whereas their expression of RANKL was comparable (Fig. 3). These data suggest a likelihood that the switch for periosteal resorption/apposition control at the mandibular periosteum is mainly through OPG rather than through RANKL. More specifically, a default amount of RANKL may be present at both resorptive and appositional regions, while the level of OPG expression induction determines which activity dominates. The current data is inadequate to depict a detailed molecular control of mandibular periosteal resorption/apposition, but following this line, future studies can focus on factors that regulate OPG expression in the mandibular periosteum.
As an established regulators for skeletal development and bone formation (Cao and Chen,2005), BMP2 was expected to express more strongly at an appositional region than a resorptive region. We found the opposite in this study, which suggests that BMP2 may also be involved in regulating periosteal bone resorption. In recent years, studies have in fact found that BMP2 can enhance osteoclastogenesis indirectly by stimulating osteoblasts or stromal cells to produce osteoclast-promoting factors, such as RANKL (Abe et al.,2000; Otsuka et al.,2003), or directly by activating osteoclast differentiation in the presence of RANKL (Jensen et al.,2009). It is hence likely that stronger expression of BMP2 at the anterior periosteal region plays an assisting role in promoting osteoclastogenesis by synergizing with RANKL.
Western blots in this study detected bands for OPG and RANKL at ∼60 kD and 50 kD, respectively, from positive controls (Santa Cruz Biotech, CA) and our samples. A molecular weight of ∼50 kD was unexpectedly higher than the predicted molecular weight for transmembrane RANKL (Wong et al.,1997), but similar findings have been reported before. Using the same antibody (Santa Cruz Biotech, CA), Dossing and Stern (2005) reported a band of ∼52 kD for RANKL, and the increased molecular weight has been attributed to RANKL glycosylation (Wong et al.,1997; Lum et al.,1999). These reports confirm that our identification of a ∼50-kD RANKL band by Western blot was specific and reliable.
Unlike mRNA expression, protein expression of OPG and RANKL examined by Western blot was not distinctly different between the two regions. While this apparent discrepancy may indicate differential transcriptional vs. translational regulations of OPG and RANKL expression at the ramal periosteum, it is more likely caused by an incomplete measurement of protein expression by Western blot used in this study. Because only proteins extracted from cell lysates were assayed, the expression of total OPG, a secreted glycoprotein, was only partially represented by the current Western blot data. The distribution of secreted/unsecreted OPG protein in periosteal cells is currently unknown, but it has been reported that normal OPG can be quickly secreted in a number of cell lines (Middleton-Hardie et al.,2006). Similarly, the current Western blotting data probably only reflected the expression level of transmembrane RANKL but not soluble RANKL. Because of these reasons, the current protein expression data was not conclusive. A future analysis of protein expression in the culture media and in in vivo tissues will clarify whether the regional variation of RANKL/OPG mRNA expression at the ramal periosteum also exists at the protein level.
The periosteum from long bones contains mesenchymal progenitor cells in the osteogenic layer (De Bari et al.,2006). Upon osteogenic induction, these cells can differentiate into osteoblasts through multiple stages. The mandibular ramal periosteal cells cultured in this study also contained such cells as exhibited by positive AP activity and strong expression of osteoblast differentiation markers (Figs. 1–3). Because of this feature, periosteal cells have been used for tissue engineering in the craniofacial region (Schmelzeisen et al.,2003; Miyamoto et al.,2004; Sakata et al.,2006). The anterior ramal surface is easy to access and has been used as a location for periosteal cell harvesting (Miyamoto et al.,2004; Sakata et al.,2006). Whether this resorptive surface is a good location to harvest periosteal cells for tissue engineer needs to be clarified. In this study, we found that during in vitro expansion, cells from the anterior surface did not differ from cells harvested from the appositional posterior surface in expressing osteogenic markers (Runx2 and OCN). However, the expression profile for RANKL/OPG ratio and BMP2 were significantly different between the two surfaces in the same way as in vivo tissues. These data suggest that cells harvested from a resorptive periosteal surface can remain osteoclastogenesis-promoting even after in vitro expansion with osteogenic media. This feature may predispose more bone resorption after transplanting them to a receiving site than using cells harvested from an appositional surface. If this speculation was further confirmed by in vivo studies, it might be a disadvantage to use periosteal cells from a resorptive surface for tissue engineering unless in vitro expansion can reverse their resorptive molecular traits.
In this study, we applied a single-dose cyclic tensile strain to cultured periosteal cells to examine whether such mechanical stimulation can change the molecular profile of periosteal cells, especially those from the anterior resorptive region. Our data showed that this dynamic tensile strain did have a tendency to stimulate osteogenic differentiation (Runx2 and OCN) of anterior cells. However, the RANKL/OPG expression ratio in cultured anterior cells also had a tendency to be increased with the application of cyclic strain, suggesting that this mechanical treatment is not effective to reverse the osteoclastogenesis-promoting feature carried by the anterior cells. While many reasons can be speculated for this result, the difference between physiological strain that the periosteal cells receive and the strain used in this study has to be considered. Physiologically, the tensile strain that displaces the mandibular periosteum, which probably also stimulates periosteal cells to be more osteogenesis-promoting, was thought to derive mainly from bone elongation (Frankenhuis-van den Heuvel et al.,1992). The exact nature of this bone-elongation strain has not been clarified, but it is probably less dynamic and more persistent than the strain used in this study. A future study using a more continuous strain with longer durations could clarify this point.
In conclusion, this study found that the unique anterior-resorption and posterior-apposition periosteal growth pattern of the mandibular ramus is likely controlled by a variation in RANKL/OPG ratio (mainly through OPG) and BMP2 expression between these two regions.