osteoclasts were observed on the surface of the alveolar bone and root (D). Three days after tooth movement, the PDL was composed of coarse arrangement of fibers and expanded blood capillaries (E). Many resorption lacunae with TRAP‐positive multinucleate osteoclasts appeared on the alveolar bone surface, while in the fibers of the PDL, many mononuclear TRAP‐positive cells were present (F). Seven days after tooth movement, fibroblasts in the PDL were increased (G). Further, on the surface of the alveolar bone, bone resorption lacunae with multinucleate TRAP‐positive osteoclasts were recognized. Mononuclear TRAP‐positive cells were decreased in comparison with these 3 days after the movement (H). The immunoreactivity of RANKL was weakly localized in cytoplasm of some fibroblasts and pericytes near the alveolar bone surface (I). One day after tooth movement, RANKL positive fibroblasts in the PDL and osteoblasts at the bone surface were increased (J). Three days after tooth movement, many RANKL positive osteoclasts and fibroblasts were observed. The immunoreactivity to RANKL of the fibroblasts become more strong (K). Seven days after movement, the immunoreactivity of RANKL was observed in the fibroblasts and osteoclasts on the alveolar bone surface, but the degree was decreased (L).
CD40, another member of the TNF superfamily, is a cell surface receptor seen in a variety of inflammatory and resident cells. Cellular responses mediated by CD40 are triggered by its counter receptor CD40L, which also belongs to the TNF gene family. It was found that CD40–CD40L interaction appears to be an active process during OTM, and that orthodontic force induces T‐cell activation (Hayashi et al., 2012). Such activation might be involved in the induction of inflammatory mediators and subsequent bone remodeling (Alhashimi et al., 2004). Indeed, it has been suggested that OPG exists in both membrane‐bound and soluble forms and that its expression is up‐regulated by CD40 stimulation.
The chemokine system
Collectively, chemokines are defined as small proteins of the cytokine family that have a broad range of activities involved in the recruitment and function of specific populations of leukocytes at the site of inflammation. Chemokine messages are decoded by specific chemokine receptors, which, once activated, regulate cytoskeletal rearrangement, integrin‐dependent adhesion, and the binding and detachment of cells from their substrate. Chemokines target all types of leukocytes and are being considered as major regulators of inflammatory processes (Silva et al., 2007). Chemokines have been identified as essential signals for the trafficking of osteoblast and osteoclast precursors, and also for the development, activity and survival of bone cells (Silva et al., 2007).
Regarding OTM, mechanical loading triggers the expression of several chemokines, which in turn create microenvironments that direct inflammatory cell migration and can influence bone formation and resorption processes. The chemokines CCL2, CCL3, CCL5, and CXCL2, which present a marked inflammatory character, were found to be highly expressed during orthodontic movement, mainly in the PDL pressure zone (Alhashimi et al., 1999; Garlet et al., 2008), which is characterized by the predominance of bone resorptive activity. Accordingly, the expression of CCL2 and CCL5 was found to be upregulated during orthodontic force application in mice PDL as being associated with the presence of TRAP positive cells (Andrade et al., 2007; Madureira et al., 2012).
Experimental studies demonstrate a pivotal role for the chemokine system in the orthodontic movement, since CCL3 deficient mice showed impairment in the amount of tooth movement and a reduced number of TRAP‐positive osteoclasts after mechanical loading (Taddei et al., 2013). It was also demonstrated that the proinflammatory and osteoclastogenic role of CCL3 is dependent on its binding to the receptor CCR1, in theory, expressed by cells of monocytic lineage and consequently potential preosteoclasts (Taddei et al., 2013). Interestingly, other chemokine receptors characteristically expressed by cells of the monocytic lineage, such as CCR2 and CCR5, are described as playing distinct roles in the OTM process. CCR2, specifically the CCR2‐CCL2 axis, is positively associated with osteoclast recruitment, bone resorption, and OTM (Taddei et al., 2012). On the other hand, the chemokine receptor CCR5 is described as a downregulator of alveolar bone resorption during orthodontic movement (Andrade et al., 2009). In this setting, a linear relation between the force and the level of CCL2 and CCL5 was shown, while higher force magnitudes did not increase the expression of such chemokines (Alikhani et al., 2015). Recently, ACKR2, a decoy receptor for CC chemokines, was demonstrated to function as a regulator of mechanically induced bone remodeling by affecting the differentiation and activity of bone cells and the availability of CC chemokines, such as CCL2 and CCL3, in the periodontal microenvironment (Lima et al., 2017). Collectively, such data suggests that different subpopulations of the monocytic lineage may be attracted to the PDL area by different chemokines, and subsequently present opposing roles in the determination of tooth movement outcome.
It is also important to consider that chemokine expression in the PDL environment can also impact soft tissue remodeling and bone formation associated with the tooth movement process. The chemokine CXCL12 was described to be highly expressed in both pressure and tension areas of mechanically challenged PDL (Garlet et al., 2008). Such chemokine is described as significantly inducing proliferation, migration, and collagen type I expression in PDL stem cells and osteoblasts (Lisignoli et al., 2006; Du et al., 2012), suggesting an anabolic role in the tooth‐movement process. The involvement of CXCL12 in OTM is reinforced by the interruption of experimental tooth movement in rats by AMD3100, an antagonist of the CXCL12 receptor, named CXCR4 (Hatano et al., 2018). Similarly, CCR5 is supposed to contribute to bone formation in response to orthodontic forces, since RUNX2 and osteocalcin levels are decreased in CCR5 deficient mice (Andrade et al., 2009).
Finally, cross regulation between cytokines and chemokines has been demonstrated in the OTM context (Andrade et al., 2009), reinforcing the existence of a complex regulatory network involved in the determination of tissue response to orthodontic forces.
Growth factors
Growth factors are usually described as molecules with the ability to stimulate or enhance cellular growth, proliferation, and differentiation; and consequently, capable of regulating a variety of cellular processes involved in development, maintenance of tissue homeostasis, and wound healing. Different types of mediators can fit in the growth factor description, including a series of cytokines, TGF‐β being the prototypic example.
TGF‐β is a pleiotropic cytokine that can regulate cell growth, differentiation, and matrix production. Generally, TGF‐β has an anabolic nature, increasing the proliferation and chemotaxis of PDL cells, and upregulating the expression of COL‐I (Matsuda et al., 1992; Sporn and Roberts, 1993; Chang et al., 2002). TGF‐β also presents anabolic properties on bone tissue, recruiting osteoblast precursors and inducing their differentiation, and enhancing the production of bone matrix proteins (Kanaan and Kanaan, 2006). TGF‐β expression was found to be increased during OTM, being observed in osteoblasts in the tension zone, and in bone‐resorbing osteoclasts in the compression zone (Kobayashi et al., 2000; Dudic et al., 2006), which suggests a broad role for this cytokine in the tooth movement process (Garlet et al., 2007). Barbieri et al. (2013) reported a significant increase in TGF‐β in GCF during OTM at the pressure side. A recent study investigated the potential application of platelet‐rich plasma, characteristically rich in growth factors such as TGF‐β, in experimental OTM in rats. However, no significant effects were observed (Akbulut et al., 2019), suggesting that the endogenous levels of molecules may be sufficient to promote an effective tooth movement.
Matrix metalloproteinases (MMPs)
These agents are zinc‐ion‐dependent proteolytic enzymes, produced by a wide variety of cells during developmental processes, inflammatory diseases, degenerative articular diseases, tumor invasion, and wound healing. These enzymes are classified into several subgroups, i.e., collagenases (MMP‐1, 8, and 13), gelatinases (MMP‐2 and 9), stromelysins, membrane‐type MMPs, and other subfamilies. Most of the MMPs are produced as pro‐enzymes, cleaved at the specific site to become a mature form, and then secreted and activated in the presence of zinc and calcium ions. The activation of MMPs is also regulated by a group of endogenous proteins named tissue inhibitors