force created by contracting muscles, which is transmitted to bone via tendon, all these three tissues can respond to the mechanical loading by producing local IGF-I or its isoform.
In skeletal muscles, insulin-like growth factor I (IGF-I) play a critical role in their formation, maintenance, and regeneration. Longitudinal exercise studies have shown that it is possible to increase muscle strength and performance without concomitant and robust changes in circulating IGF-I. Such studies indicating that the effect of exercise on skeletal muscles is mediated via paracrine/autocrine IGF-I rather than endocrine IGF-I (Frystyk, 2010). Goldspink (Goldspink, 1999) showed that loading increases the production of IGF-I in muscle cells, functioning in an autocrine/paracrine mode, is an important mediator of skeletal muscle adaptation being sensitive to increases in loading (Adams, 2002).
In human tendon, which resembles the PDL in its function, it can be demonstrated that an increase in the interstitial concentration of IGF-I and its binding proteins takes place after exercise. The increase in IGF-I expression in tendon includes the isoform that has so far been thought only to exist in skeletal muscle (mechano-growth factor) (Kjaer et al., 2009). In rats, it was suggested that the IGF-I and its splice variant mechano-growth factor (MGF) could be involved in collagen synthesis in tendon in response to mechanical loading (Olesen et al., 2006).
The transmitted mechanical stimulation to bone leads to increased bone formation and plays an essential role in maintaining skeletal integrity. Growth factors and osteocytes, which act as mechanosensors, play a key role during the bone formation after mechanical stimulation. Recently, Reijnders et al. (2007) have found that IGF-I mRNA is upregulated within endocortical osteocytes of the shaft of rat tibia 6 h after mechanical loading. They suggested that IGF-I, which is located in osteocytes, is involved in the translation of mechanical stimuli into bone formation. It has been also demonstrated that rat osteoblasts respond to the mechanical forces which may regulate their activities indirectly by promoting the autocrine effect of IGF-1 (Xian et al., 2007).
The ability of IGF-I to translate the mechanical stimuli into bone formation was investigated and verified in many experimental and clinical studies.
Distraction osteogenesis is currently a standard method of bone lengthening (Choi et al., 2002). In distraction osteogenesis studies, gene expression of IGF-I was detected during distraction (Farhadieh et al., 1999; Yates et al., 2002).
The application of tensile forces to the mid sagittal cranial suture during the growth period cause an increase in the production of IGF-I mRNA and IGF-IR mRNA in osteoblast-like and fibroblastic cells (Hirukaw et al., 2005).
Hajjar et al. (2002) observed that the expression of IGFs increases in condylar cartilage in response to application of propulsive appliances.
In orthodontics, Wescott et al. have applied a 12 % uni-axial cyclic tensile strain to cultured human PDL cells and analyzed the differential expression of 78 genes, 19 genes, including IGF-I, show differential expression. The treated/control (T/C) ratio of IGF-I after 12 hrs was greater than of ± 2 (2.46) but not significant (Wescott et al., 2007). Rath-Deschner et al. have found that IGF-I expression is significantly increased, when cells were subjected to low continuous tensile strain for 4 h ( Rath-Deschner et al., 2009). Saggese et al. found a fivefold increase of IGF-I in the cervicular fluid after insertion of an orthodontic appliance to distalize canines in patients aged between 8 and 15 years (Saggese et al., 2005). Toia et al. found that IGF-I increases in the cervicular fluid as early as 4hrs after insertion of fixed appliances (Toia et al., 2005). Therefore, this study indicates that IGF-I and its signals involved in the mechanotransduction processes during the tooth movement.
In this study, the character of IGF-I over time was observed in the PDL and found that IGF-I-positive cells were significantly increased at the tension side in a time dependent manner. It was also recognized that this increase correlated with an increase in the total number of PDL cells over time at the same side. The both IGF-I-positive cells and the total number of the PDL cells reach their peak points after 24 hrs.
It has been observed that the mitotic activity of PDL cells increases markedly on the tension side from 24 to 36 hrs after the initiation of tooth movement (Macapanpan et
al., 1954). This leads to increase the number of periodontal ligament cells, which can be increased by stimulating cell proliferation or by inhibiting apoptosis (Suri and Taneja, 2008). These cell functions can be affected through IGF-I production. IGF-I stimulates proliferation of PDL fibroblast (Palioto et al., 2004), cementoblasts (Grzesik and Narayanan, 2002) and osteoblasts (Wergedal et al., 1990).
The autocrine/paracrine secreted IGF-I is also an important component of the response to injury. Multiple studies in several different animal models have shown that IGF-I is synthesized after injury by the cell types that account for tissue regeneration. This synthesis is necessary for normal tissue repair (Clemmons and Maile 2005), defends against cellular stress and delays the onset of the apoptosis (Kumasheva and Houghton, 2006). The antiapoptotic action of IGFs was recently also observed in PDL cells that respond to IGF-I by up-regulation of anti-apoptotic pathways (Han and Ammar, 2003). Therefore, one can presume that the up-regulation of IGF-I on the tension side can increase the cell number of PDL through its impacts on the proliferation and the apoptosis of the PDL cells during the tooth movement in a time-dependent manner.
The mean values of immunopositive cells for all examined factors reach the peak point after 24 hrs compared with those of the control teeth. These results are in accordance with those described by other researchers for other factors. Transforming growth factor-β1 (TGF-β1) regulates cell proliferation, differentiation, motility and apoptosis (Kanaan and Kanaan, 2006). Osteoprotegerin (OPG) inhibits the differentiation and stimulates the apoptosis of the osteoclasts (Tyrovola et al., 2008).
These both factors were observed with significant increase in stretched cells on the bone surface after 24 hrs from force application and became more intense after 48 hrs of force application (Kobayashi et al., 2000).
The expression of epidermal growth factor (EGF) and its receptor, which play an important role in bone formation, were detected in periodontal tissues after 24 hours and 168 hours of tooth movement. The expressions of EGF and EGFR were increased
in periodontal tissues at 168 hrs higher than those at 24 hrs and at tension side higher than those at pressure side at the same time (Gao et al., 2002).
The expression patterns of Ki-67, a cellular marker for proliferation and osteoblast precursors (Scholzen and Gerdes et al., 2000), and Runx2, an essential transcription factor for osteoblast differentiation and chondrocyte maturation (Komori, 2011), were investigated at 3 and 24 hours after appliance insertion. The results showed that the expression of Ki-67 and Runx2 increase in the tension areas after 24 hours of force application (Brooks et al., 2009).
Recently, the gingival crevicular fluid (GCF), which can be collected from the gingival crevice surrounding the teeth, was used to determine possible changes in its constituent in response to various pathological and physiological alterations in the periodontium (Lamster et al., 2007). The presence/expression of regulatory proteins in the GCF has been examined to illustrate the involvement of these proteins in periodontal remodeling provoked by orthodontic stimuli. The most consistent result was a peak of cytokine levels at 24 h (Ren and Vissink, 2008). For example, Nishijima et al., have examined the secretion of RANKL and OPG from hPDL cells at the distal cervical margins of the experimental and control teeth 0, 1, 24, and 168 hrs after the retracting force was applied. After 24 hrs, the levels of RANKL were significantly higher and the levels of OPG were significantly lower compared to the control teeth.
There were no such significant differences at 0, 1, or 168 hrs (Nishijima et al., 2006).
All these results support the statement that cellular and tissue reactions, including IGF-I synthesis, start in the initial phase (1-2 days) of tooth movement, immediately after force application. The complex processes of recruitment of osteoclast and osteoblast progenitors begin in this early phase (Krishnan and Davidovitch, 2006).
The human PDL cells represent a heterogeneous cell population including stem cells (Lekic et al., 2001; Silvério et al., 2010) and have the potential to differentiate into various phenotypes, including osteoblasts (Gay et al., 2007) and cementoblasts (Seo et al., 2004). IGF-I induces many differentiated functions, such as production of
collagen and matrix apposition (Grzesik and Narayanan, 2002; Minuto et al., 2005). It is known that procollagen type I is an indicator of the early stage of bone formation.
The recombinant IGF-I can raise the serum levels of procollagen type I significantly (Zofkova, 2003). Therefore, it is assumed that IGF-I activates especially the early stages of osteoblastic formation in the PDL.
In periodontal regeneration, the reestablishment of the PDL is required together with corresponding cementum and supporting alveolar bone. Thus, agents that promote PDL cells proliferation and migration as well as collagen biosynthesis would appear to be mediators for enhancing new PDL formation (Raja et al., 2009). Recently, in two in vitro studies, IGF-I alone or in combination with other growth factors promoted osteogenesis of the PDL cells. It can elevate the osteoblastic markers such alkaline phosphatase (ALP) and ostetocalcin (OCN) (Chen et al., 2009; Li et al., 2011). IGFs regulate, in diverse patterns, the differentiation functions of both osteoblasts and osteoclasts (Conover, 2000). This can accelerate the differentiation of osteoclasts and its disappearance in the tension side after force application (disto-coronal areas), which tend to contain more osteoclasts in the PDL of untreated teeth due to the physiological distal drift of the molars in the rats.
Therefore, one can speculate that the up-regulation of IGF-I could influence the differentiation of the PDL cells at the tension side.
As previously mentioned in the introduction, there are four essential interrelated steps in the transduction of mechanical signals by tissues: sensing the mechanical signal by the cells, transduction of this mechanical signal into one that is biochemical, transmission of the biochemical signal to the effector cells, and the effectors cell response (Wise and King, 2008).
After force application both matrix strain and fluid flow in the PDL and the bone cause deformation of cells. Through integrin signalling and other transduction pathways, many mediators are produced leading to activate several types of cells (Hennemann et al., 2008).
Ajubi et al. investigated the signal transduction pathways in osteocytes which were subjected to pulsating fluid flow (PFF). They found that PFF raises intracellular Ca2+. Ca2+ and protein kinase C then stimulate phospholipase A2 activity, arachidonic acid production, and ultimately prostaglandin E2 (PGE2) release (Ajubi et al., 1999; Wise and King, 2008).
Studies which were performed in gingival crevicular fluid (GCF) during tooth movement showed that the concentration of PGE2 in GCF increased significantly and reaches peak point at 24th hour and generally was higher in tension sides than at the compression sides (Yao et al., 2003; Dudic et al., 2006).
Release of PGE2 is a prominent load-induced response of osteoblast-like cells. PGE2 is produced by osteoblasts in response to physiological stress, growth factors, hormones, trauma or inflammatory cytokines and induces cAMP-dependent IGF-I expression by osteoblasts (Papachroni et al., 2009).
In vitro studies showed that PGE2 increases rapidly at 5min after loading of teeth and led to an increase of intracellular cyclic adenosine monophosphate (cAMP) which peaks at 15 min. (Meikle, 2006).
The increased intracellular cAMP activates protein kinase A (PKA), which stimulates IGF-I gene induction (McCarthy and Centrella, 2001). In addition, mechanical interactions between integrins and their matrix/environment mediate increases in intracellular Ca2+ levels and activate mitogen-activated protein (MAP) kinase cascades. This leads to the activation of the activator protein 1 (AP-1) that is necessary for a pro-growth response. The pro-bone growth response involves up-regulation of the genes c-fos, osteocalcin, cyclooxygenase, and IGF-I (Iqbal and Zaidi, 2005).
and thus can man better understand the mechanism of IGF-I up regulation after tooth movement as response to up regulation of other factors such as PGE2.
In this study, immunoreactivity for IGF-IR was generally more pronounced than that for its ligand IGF-I. The biological actions of IGF-I are predominantly mediated by the IGF-IR (LeRoith 2000; Romano, 2003; Annunziata et al., 2011).
Recently, it has been reported, that growth factor receptors can be also activated by integrins in the absence of the growth factor ligands, due to the interaction between integrins and growth factor receptors (Clemmons and Maile, 2003; Beattie et al., 2010). The crosstalk between integrins and growth factor receptors in multiple cell types may be the result of coclustering of these receptors on the surface of the cell in focal adhesions or in association with the actin cytoskeleton (Eliceiri, 2001, Beattie et al., 2010). For example, the occupation of the αVβ3 integrin receptor with ECM proteins induces IGF-I-stimulated IGF-IR phosphorylation. Conversely, the presence of the αVβ3-specific disintegrin echistatin inhibits the IGF-I-stimulated IGF-IR activation (Kim et al., 2007). In this context, Kapur et al. found that echistatin reduced not only the basal and shear stress-induced TE85 cell (osteosarcoma cell) proliferation but also completely abolished the increase in cell proliferation induced by IGF-I alone as well as that by the combination treatment. This suggests that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR (Kapur et al., 2005).
Insulin receptor substrates (IRS) is a main target molecule of insulin/IGF-1 receptor signaling and plays important roles in maintaining normal bone turn-over (Ogata and Kawaguchi, 2008). When the concentrations of IRS1 are high, the signal is mitogenic and anti-apoptotic (Valentinis and Baserga, 2001). IRS1 deficient mice exhibited severe low turnover osteopenia with decreased bone anabolic and catabolic action (Akune, 2004).
Tyrosine phosphorylation of IRS1 by the IGF-I receptor tyrosine kinase leads to activation of pathways such PI3 and extracellular signal-regulated kinase 1/2 (ERK1/2). The first pathway protects from apoptosis by blocking the pro-apoptotic proteins and inducing the expression of antiapoptotic proteins. The second pathway, ERK1/2, is required for optimal stimulation of cell division (proliferation) and gene expression (Jones and Clemmons, 1995; Mauro and Surmacz, 2003; Yamagushi et al, 2005). The application of orthodontic loading within the same time interval and under identical experimental circumstances induced a significant increase of ERK1/2-positive PDL cells at the tension side (Kawarizadeh et al., 2005, Pavlidis et al., 2009).
It most kept in mind that ERK1/2 can be activated by other molecules like integrin receptors (Iqbal and Zaidi, 2005; Meikle, 2006).
However, the ability of cells to respond to the autocrine/ paracrine secreted IGF-I is dependent not only on the amount of growth factor that is secreted, but also on the abundance of other proteins in the extracellular environment especially IGFBPs.
IGFBPs are a family of secreted proteins that bind IGF with high affinities that are equal to or greater than those of the IGF-IR. In the extracellular environment the most, if not all, of IGFs are bound to specific, high-affinity IGFBPs (Duan and Xu, 2005).
They can suppress or enhance the cellular effects of IGFI (Yu and Rohan, 2000; Firth and Baxter, 2002). Anyway, the observed up-regulation of the IGF-IR and IRS1 in this study strengthens the view that the biological action of IGF-I on PDL cells was stimulated under this condition of force application.
At the pressure side, it was observed that all factors are significantly down regulated in the 4 hrs groups, but not in 24 or 48 hrs groups, where the differences between the groups were no more significant. This may be caused by virtue that PDL cells subjected to tensional and compressive forces are able to perceive two different forms of mechanical stimuli and responded in a different manner (He et al., 2004).
Other interpretation for down regulation of IGF-I at the pressure side could due to the negative strain that induced within the PDL at the pressure side of the root (Hennemann et al., 2008) and, consequently, the fibers are relaxed. This leads to unloading of the PDL and alveolar bone (Melsen , 1999, 2001; Binderman et al., 2002). The unloading reduces canalicular fluid flow and induces the apoptosis of osteocytes and osteoblasts, which attracts osteoclasts to the pressure side followed by bone resorption subsequently (Burger et al., 2003; Aguirre et al., 2006; Dufour et al., 2007; Hennemann et al., 2008). Unloading could also inhibit the proliferation and differentiation of osteoprogenitor cells in vitro (Kostenuik et al., 1997). Moreover, skeletal unloading induces resistance to IGF-I on bone formation and inhibits the activation of the IGF-I signaling pathway (Sakata et al., 2003, 2004).
Clinically, skeletal unloading during space flight and prolonged bed rest induces osteopenia (Bikle and Hallaoran, 1999).
Thus, one can assume that there is a relation between the unloading of PDL cells, the initial decrease of IGF-I-positive cells, and the decrease of the total cell number at the pressure side.
The positive PDL-cells at the pressure side decreased in the 4 hrs group and augmented subsequently in 24 and 48 hrs groups. This could be interpreted as result of the remodeling activity of PDL tissue and alveolar bone at the pressure side that degrade to create space for the moving tooth while new PDL tissue is simultaneously formed to maintain the attachment (Hennemann et al. 2008).
At the cellular level, PDL cells in the pressure side become a round shape in the first hours of treatment and regain a normal shape 2 or 3 days later (Kirshnan and Davidovitch, 2006). In this regard, cells which are rounded up show catabolic changes whereas flattened cells (under tension) have anabolic effects (Roberts-Harry and Sandy, 2004).
In the present study, the effect of the force magnitude on the positive cells number was only significant between the groups, where 0.1N and 0.5N were applied for 4 hrs and only for IGF-IR. The results for IGF-I and IRS1 showed no significant differences between applied forces but tend to be negative dependent on the force magnitude. These results could indicate a relation between the expression of these molecules, especially IGF-IR, and the magnitude of the applied forces.
In the past 70 years, the concept of optimal force has changed considerably. According to Schwarz, who defined optimal continuous force as „the force leading to a change in tissue pressure that approximated the capillary vessels‟, forces below the optimal level cause no reaction in the periodontal ligament. Forces exceeding the optimal level would lead to areas of tissue necrosis, preventing frontal bone resorption. Tooth movement would thus be delayed until undermining resorption had eliminated the necrotic tissue obstacle (Schwarz, 1932). Oppenheim and Reitan modified this
definition and advocated the use of the lightest force which is capable to achieve the tooth movement (Oppenheim, 1942; Reitan, 1967). The current concept of optimal force is based on the hypothesis, that a force of a certain magnitude and temporal characteristics would be capable of producing a maximum rate of tooth movement without tissue damage and with maximum patient comfort. The optimal force for tooth movement may differ for each tooth and for each individual patient (Ren et al., 2003).
There are many reports about the effect of force magnitude on tooth movement. King et al. demonstrated that the effective tooth movement of rat molars ranges from 20 g to 40 g and its velocity does not increase over 40 g (King et al., 1991). Gonzales et al.
found that the light forces produce more tooth movement and less root resorption compared with heavier forces (Gonzales et al., 2007).
Considering the orthodontic tooth movement as the result of biological reactions to externally applied mechanical stimuli, the application of 0.1 N may cause greater biologic activity of IGF-I-signalling than the other forces, which can subsequently lead to cellular responses aiming at adaptation to the applied forces.