3. Molecular mechanism of orthodontic tooth movement

Although there are several models proposed to explain the events of orthodontic tooth movement, no single model could directly and comprehensively explain this process. The evidence from histological and animal studies has shown that this complex biological process is initiated from the application of mechanical forces onto the orthodontic appliances, which converts into the biological signals to stimulate mechanosensitive cells. (Figure 1) [22] Literatures showed that orthodontic force application induced physiologic adaptation of alveolar bone with small magnitude of reversible injury to periodontium [23]. Significant evidence suggests that when mechanical loading forces are relayed from the orthodontic appliances to the PDL and bone tissues, the mechanoreceptor cells percept the loading forces as shear stress and strain [24] as the tooth shifts its position in the PDL space resulting in compression and tension areas in PDL and bone tissues [25].

The sequence of biological events after loading of orthodontic force occurs as (1) fluid flow changes and matrix strain (Figure 2); (2) strain on mechanoreceptor cells (Figure 2); (3) cell activation (Figure 3); and (4) tissue remodeling leading to tooth movement (Figure 4) [15].

Figure 1. Illustration of cellular events of periodontal ligament and alveolar bone at non-loading state. Blood vessels and periodontal fibroblasts reside in between the periodontal ligament collagen fibers. Inactive osteoblasts line along the alveolar bone surface and quiescent osteocytes reside in their bony lacunae. Modified from Hatch [25].

The mechanoreceptor cells in periodontal tissue include osteocytes and bone lining osteoblasts in alveolar bone and fibroblasts in PDL. The final result as tissue remodeling occurs in both mineralized and non-mineralized ECM during the tooth movement [26]. Recent studies have indicated that osteocytes are capable of sensing strain in their bone lacunae following mechanical loading of the bone [21]. The mechanism of how osteocytes sense, transfer, and respond to mechanical strain remains unclear. Osteocyte processes have been shown to utilize integrins, gap junctions and ion channels to respond to mechanosensing external physical stimuli [27, 28]. Fluid flow-induced shear stress is the strain resulted from an immediate change in fluid flow in the lacunarcanalicular system leading to an increasing strain on the osteocytes. This shear stress can amplify the mechanical signals to the osteocytes [14, 29]. Several proteins such as integrins, connexin 43, osteopontin, and vitronectin, and several transcriptional factors such as c-Fos expression in the osteocytes are affected by loading forces [30–32]. In addition, the reduced number of primary cilia of osteocytes could affect their secretion of prostaglandins (PGs) and increased cyclooxygenase-2 (COX2) and RANKL/OPG ratio in osteocytes in response to fluid flow shear stress [33, 34]. Recent studies showed that osteocytes can induce both anabolic and catabolic bone signals in response to loading [35–37], yet the mechanism of how osteocytes switch from catabolic activity to anabolic activity is unclear. Under compression, osteocytes undergo apoptosis and are coupled with bone resorption [19, 38]. However, fluid flow shear stress may induce osteocytes to secrete anabolic bone proteins such as prostaglandin-E2 (PGE2) or nitric oxide (NO) [39, 40]. Several recent evidence demonstrated the significance of osteocytes during osteoclast differentiation and

activation [41–43]. The osteocyte ablation in vivo caused a significant reduction in osteoclastogenesis and osteoclastic activity under loading forces, suggesting the important roles of osteocytes during orthodontic tooth movement [44]. Increased evidence supported the close association between osteocytes and osteoclasts during tooth movement. Experimental tooth movement in mice demonstrated increased expression of osteopontin [45], matrix extracellular phosphoglycoprotein (MEPE) [46], and receptor activator of nuclear factor-kB ligand (RANKL) [43, 47] in osteocytes. These proteins play important roles in osteoclastic activity and osteoclastogenesis because deficiency of these proteins results in significant reduction or absence of the osteoclasts and increased bone mass in the animals [43, 48]. Osteocyte apoptosis occurred abundantly on the compression side of tooth movement in 1 day after loading, and then an increased number of osteoclasts were observed until day 3, resulting increased tooth movement by day 10 [49]. It is speculated that apoptotic osteocytes may release signaling proteins such as RANKL and interleukin (IL), to osteoclast precursors, and initiate osteoclastogenesis. In contrast, when subjected to fluid flow sheer stress, osteocytes secrete NO and PGE2, which these proteins have potent, anabolic, and direct effects on osteoblasts [40, 50, 51]. PGE2 expression increased in loaded bone tissue [52]. NO secreted from osteocytes promotes osteoblast differentiation, and

osteocytes. Modified from Hatch [25].

Figure 2. Initial cellular events in periodontal ligament after force loading during tooth movement. The blood vessels are squeezed then local hypoxia and fluid flow change are initiated from the loading force. The mechanical strain affects the periodontal fibroblasts and osteoblasts in the periodontal ligament space. The strain creates fluid flow shear stress and strain on the osteocytes in their bone lacunae. The mechanical strain induces secretion of inflammatory cytokines and biological signaling mediators including interleukins, prostaglandins, tumor necrosis factors, nitric oxide, growth factors, proteinases and cell differentiation factors. These mediators, in turn, activate the periodontal fibroblasts, osteoblasts and

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Figure 2. Initial cellular events in periodontal ligament after force loading during tooth movement. The blood vessels are squeezed then local hypoxia and fluid flow change are initiated from the loading force. The mechanical strain affects the periodontal fibroblasts and osteoblasts in the periodontal ligament space. The strain creates fluid flow shear stress and strain on the osteocytes in their bone lacunae. The mechanical strain induces secretion of inflammatory cytokines and biological signaling mediators including interleukins, prostaglandins, tumor necrosis factors, nitric oxide, growth factors, proteinases and cell differentiation factors. These mediators, in turn, activate the periodontal fibroblasts, osteoblasts and osteocytes. Modified from Hatch [25].

The mechanoreceptor cells in periodontal tissue include osteocytes and bone lining osteoblasts in alveolar bone and fibroblasts in PDL. The final result as tissue remodeling occurs in both mineralized and non-mineralized ECM during the tooth movement [26]. Recent studies have indicated that osteocytes are capable of sensing strain in their bone lacunae following mechanical loading of the bone [21]. The mechanism of how osteocytes sense, transfer, and respond to mechanical strain remains unclear. Osteocyte processes have been shown to utilize integrins, gap junctions and ion channels to respond to mechanosensing external physical stimuli [27, 28]. Fluid flow-induced shear stress is the strain resulted from an immediate change in fluid flow in the lacunarcanalicular system leading to an increasing strain on the osteocytes. This shear stress can amplify the mechanical signals to the osteocytes [14, 29]. Several proteins such as integrins, connexin 43, osteopontin, and vitronectin, and several transcriptional factors such as c-Fos expression in the osteocytes are affected by loading forces [30–32]. In addition, the reduced number of primary cilia of osteocytes could affect their secretion of prostaglandins (PGs) and increased cyclooxygenase-2 (COX2) and RANKL/OPG ratio in osteocytes in response to fluid flow shear stress [33, 34]. Recent studies showed that osteocytes can induce both anabolic and catabolic bone signals in response to loading [35–37], yet the mechanism of how osteocytes switch from catabolic activity to anabolic activity is unclear. Under compression, osteocytes undergo apoptosis and are coupled with bone resorption [19, 38]. However, fluid flow shear stress may induce osteocytes to secrete anabolic bone proteins such as prostaglandin-E2 (PGE2) or nitric oxide (NO) [39, 40]. Several recent evidence demonstrated the significance of osteocytes during osteoclast differentiation and

Figure 1. Illustration of cellular events of periodontal ligament and alveolar bone at non-loading state. Blood vessels and periodontal fibroblasts reside in between the periodontal ligament collagen fibers. Inactive osteoblasts line along the

alveolar bone surface and quiescent osteocytes reside in their bony lacunae. Modified from Hatch [25].

54 Current Approaches in Orthodontics

activation [41–43]. The osteocyte ablation in vivo caused a significant reduction in osteoclastogenesis and osteoclastic activity under loading forces, suggesting the important roles of osteocytes during orthodontic tooth movement [44]. Increased evidence supported the close association between osteocytes and osteoclasts during tooth movement. Experimental tooth movement in mice demonstrated increased expression of osteopontin [45], matrix extracellular phosphoglycoprotein (MEPE) [46], and receptor activator of nuclear factor-kB ligand (RANKL) [43, 47] in osteocytes. These proteins play important roles in osteoclastic activity and osteoclastogenesis because deficiency of these proteins results in significant reduction or absence of the osteoclasts and increased bone mass in the animals [43, 48]. Osteocyte apoptosis occurred abundantly on the compression side of tooth movement in 1 day after loading, and then an increased number of osteoclasts were observed until day 3, resulting increased tooth movement by day 10 [49]. It is speculated that apoptotic osteocytes may release signaling proteins such as RANKL and interleukin (IL), to osteoclast precursors, and initiate osteoclastogenesis. In contrast, when subjected to fluid flow sheer stress, osteocytes secrete NO and PGE2, which these proteins have potent, anabolic, and direct effects on osteoblasts [40, 50, 51]. PGE2 expression increased in loaded bone tissue [52]. NO secreted from osteocytes promotes osteoblast differentiation, and

Figure 3. Intermediate cellular events in periodontal ligament during tooth movement. The blood vessels dilate due to the response to the released mediators and cytokines. The activated fibroblasts, osteoblasts and osteocytes are ready to secrete M-CSF and RANKL to activate preosteoclasts from blood and bone marrow. In addition, the activated osteoblasts release OPG to act as competitive decoys for RANKL. The PDL fibroblasts release MMPs to degrade collagen fibers in the periodontal ligaments. Modified from Hatch [25].

tooth movement. During the process, inflammatory cytokine such as IL-1β, PGE2, tumor necrosis factor-alpha (TNF-α) and NO are secreted from preosteoblast in PDL and osteocytes in bone lacunae during the orthodontic tooth movement [59, 65, 66]. Compression is associated with elevated COX-2 which catalyzes production of PG, including PGE2, from arachidonic acid [67, 68]. Administration of PGE2 into alveolar bone of mice induces both osteoclasts and osteoblasts [26]. During orthodontic tooth movement, pain sensation occurs and, coincidentally, substance P and calcitonin gene related peptide (CGRP) are induced to be secreted during the tooth movement. These neuropeptides can enhance cellular secretion of inflammatory cytokine and in turn increase vasodilation and permeability of surrounding blood vessels [69–72]. Several evidence showed that the inhibition of inflammation hindered tooth movement [73, 74], while inflammation in the alveolar bone promoted tooth movement [75, 76].

Figure 4. Late cellular events in periodontal ligament and alveolar bone front during tooth movement. The activated osteoclast is derived from the fusion of preosteoclasts, creates ruffle border to seal the bone surface area and releases MMP9, TRAP and acid to resorb bone matrix and minerals. Apoptotic osteocytes also release the biomolecules and mediators to activate osteoclast recruitment for bone resorption leading to tooth movement. Modified from Hatch [25].

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Osteoclasts are the major key cells that play significant roles during tooth movement. Osteoclasts are multinucleated giant cells which are formed by the fusion of mononucleated osteoclast precursors derived from hematopoietic origin and function to resorb the alveolar bone during tooth movement. The osteoclast progenitor cells require macrophage colony stimulating factors (M-CSF) for their proliferation and survival. M-CSF is a secreted cytokine by osteoblasts and affects osteoclast progenitors. The RANK/RANKL/OPG system has been a crucial mechanism in osteoclastogenesis during bone resorption and tooth movement [77–79]. Receptor activator for nuclear factor κB (RANK) is a transmembrane protein and a member of tumor necrosis factor

plays an important role in bone formation during loading [40, 53]. NO can influence bone mass and simultaneously inhibit osteoclast activity [54]. Increased NO production by osteocytes after mechanical stimulation by fluid flow modulates apoptosis-related gene expression suggesting that NO maintains osteocyte viability [55].

Beside osteocytes, preosteoblasts are also responsive to mechanical force. Mechanical force loading triggers several cell signaling pathways in osteoblasts such as calcium (Ca2+), NO, IL1β and adenosine triphosphate (ATP) in a short period of time [24, 56, 57]. NO and IL are potent mediators secreted during orthodontic tooth movement [58, 59]. Preosteoblast differentiation can be induced on the tension side of tooth movement via integrin/focal adhesion kinase signaling and Ca2+ channels [60, 61]. Fluid shear stress can trigger Ca2+ signaling pathway and promotes ATP release, PGE2 secretion and proliferation of osteoblasts [24, 57]. While on the compression side, reduced blood flow in PDL and localized hypoxia occurs. The reduction in O2 tension stabilizes hypoxia inducible factor-1 (HIF-1), a transcription factor that activates vascular endothelial growth factor (VEGF) and RANKL expression in PDL fibroblasts and osteoblasts leading to osteoclast differentiation and favoring bone resorption in areas of compression [62–64]. As mentioned above, inflammatory cascade is important for orthodontic Advances in Orthodontic Tooth Movement: Gene Therapy and Molecular Biology Aspect http://dx.doi.org/10.5772/intechopen.80287 57

Figure 4. Late cellular events in periodontal ligament and alveolar bone front during tooth movement. The activated osteoclast is derived from the fusion of preosteoclasts, creates ruffle border to seal the bone surface area and releases MMP9, TRAP and acid to resorb bone matrix and minerals. Apoptotic osteocytes also release the biomolecules and mediators to activate osteoclast recruitment for bone resorption leading to tooth movement. Modified from Hatch [25].

tooth movement. During the process, inflammatory cytokine such as IL-1β, PGE2, tumor necrosis factor-alpha (TNF-α) and NO are secreted from preosteoblast in PDL and osteocytes in bone lacunae during the orthodontic tooth movement [59, 65, 66]. Compression is associated with elevated COX-2 which catalyzes production of PG, including PGE2, from arachidonic acid [67, 68]. Administration of PGE2 into alveolar bone of mice induces both osteoclasts and osteoblasts [26]. During orthodontic tooth movement, pain sensation occurs and, coincidentally, substance P and calcitonin gene related peptide (CGRP) are induced to be secreted during the tooth movement. These neuropeptides can enhance cellular secretion of inflammatory cytokine and in turn increase vasodilation and permeability of surrounding blood vessels [69–72]. Several evidence showed that the inhibition of inflammation hindered tooth movement [73, 74], while inflammation in the alveolar bone promoted tooth movement [75, 76].

plays an important role in bone formation during loading [40, 53]. NO can influence bone mass and simultaneously inhibit osteoclast activity [54]. Increased NO production by osteocytes after mechanical stimulation by fluid flow modulates apoptosis-related gene expression suggesting

Figure 3. Intermediate cellular events in periodontal ligament during tooth movement. The blood vessels dilate due to the response to the released mediators and cytokines. The activated fibroblasts, osteoblasts and osteocytes are ready to secrete M-CSF and RANKL to activate preosteoclasts from blood and bone marrow. In addition, the activated osteoblasts release OPG to act as competitive decoys for RANKL. The PDL fibroblasts release MMPs to degrade collagen fibers in the

Beside osteocytes, preosteoblasts are also responsive to mechanical force. Mechanical force loading triggers several cell signaling pathways in osteoblasts such as calcium (Ca2+), NO, IL1β and adenosine triphosphate (ATP) in a short period of time [24, 56, 57]. NO and IL are potent mediators secreted during orthodontic tooth movement [58, 59]. Preosteoblast differentiation can be induced on the tension side of tooth movement via integrin/focal adhesion kinase signaling and Ca2+ channels [60, 61]. Fluid shear stress can trigger Ca2+ signaling pathway and promotes ATP release, PGE2 secretion and proliferation of osteoblasts [24, 57]. While on the compression side, reduced blood flow in PDL and localized hypoxia occurs. The reduction in O2 tension stabilizes hypoxia inducible factor-1 (HIF-1), a transcription factor that activates vascular endothelial growth factor (VEGF) and RANKL expression in PDL fibroblasts and osteoblasts leading to osteoclast differentiation and favoring bone resorption in areas of compression [62–64]. As mentioned above, inflammatory cascade is important for orthodontic

that NO maintains osteocyte viability [55].

periodontal ligaments. Modified from Hatch [25].

56 Current Approaches in Orthodontics

Osteoclasts are the major key cells that play significant roles during tooth movement. Osteoclasts are multinucleated giant cells which are formed by the fusion of mononucleated osteoclast precursors derived from hematopoietic origin and function to resorb the alveolar bone during tooth movement. The osteoclast progenitor cells require macrophage colony stimulating factors (M-CSF) for their proliferation and survival. M-CSF is a secreted cytokine by osteoblasts and affects osteoclast progenitors. The RANK/RANKL/OPG system has been a crucial mechanism in osteoclastogenesis during bone resorption and tooth movement [77–79]. Receptor activator for nuclear factor κB (RANK) is a transmembrane protein and a member of tumor necrosis factor receptor family that is expressed on osteoclastic precursors, preosteoclasts and osteoclasts. Receptor activator for nuclear factor κB ligand (RANKL) is a transmembrane protein and is a member of the tumor necrosis factor superfamily that is expressed on preosteoblasts, osteoblasts and osteocytes [80]. RANK is the receptor for RANKL and the binding between both of them stimulates the differentiation of preosteoclasts into mature osteoclasts. Osteoprotegerin (OPG) is a soluble extracellular tumor necrosis receptor protein that is secreted by preosteoblasts and osteoblasts. OPG is a decoy receptor for RANKL in regulating bone metabolism and inhibiting osteoclastogenesis and bone resorption. RANKL/OPG ratio is an important determinant of bone mass and skeletal integrity and also an indicator for the osteoclast function [78, 79]. Increased evidence demonstrated the direct association of tooth movement and activities of osteoclasts. Accelerated osteoclast resorption in alveolar bone of OPG deficient mice was observed during tooth movement [81] while inhibition of RANKL or deletion of RANKL in mice resulted in suppression of tooth movement [47]. In addition, local administration of M-CSF resulted in modulation of rate of tooth movement in animals [82].

of these proteins once administered in human body is short and may not reach therapeutic level [95]. Gene therapy is a therapeutic approach that uses genes to treat or prevent diseases. Gene therapy is designed to introduce nucleotides into the cells to compensate for mutated genes or to restore the normal protein. If a mutation causes a crucial protein to be defective or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. After integration of the genes that encoded the target protein into the patient's genetic machinery, gene therapy can allow the body to produce the required protein constantly so the level of protein will be constantly high at therapeutic level [96]. The concept of gene therapy includes cloning of selected DNA/RNA fragments into a delivery system in order to administer into the host or patient. The delivery system could be viral vectors or nonviral vectors such as liposomes, peptides, polymer particles, gene gun and electric perforation [97]. The clinical application of gene therapy can be achieved with in vivo or ex vivo approaches. The in vivo gene therapy will include injection of vectors into the patient directly while the ex vivo approach includes the introduction of vector into the cells then the transfected cells are

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Recently gene therapy has been approved to be implemented in medicine. The U.S. Food and Drug Administration (FDA) regulates all gene therapy products in the United States and oversees research in this area. In medicine, the FDA recently approved gene therapy for the treatment of some types of leukemia and inherited blindness [101]. Several experiments of gene therapy in dentistry involved orofacial pain, squamous cell carcinoma, tooth and bone

The gene therapy experiments in orthodontic treatment are still limited to cell cultures or animal experiments [103]. The purposes of previous gene therapy in orthodontic treatment were to investigate the possibility of acceleration of tooth movement or reduction of root resorption by modification of osteoclast differentiation factors such as RANKL or OPG [104–109]. The first attempt for gene therapy in orthodontic treatment aimed to transfer OPG gene into periodontal tissue to reduce osteoclast activity and inhibit tooth movement. The gene transfer approach using a hemagglutinating virus of Japan (HVJ) envelope vector carrying mouse OPG messenger RNA (mRNA) was performed in rats for 21 days of tooth movement. The vector solution was administered into rat's palatal gingiva by infiltration injection. The result showed that local OPG gene transfer reduced the number of osteoclasts and decreased tooth movement by 50% in rats in the experimental group compared to the ones in the control group. The effect of OPG gene transfer was local and did not affect bone mineral density of tibia of the animals [105]. The same group of investigators performed another experiment using the same system to transfer mouse RANKL mRNA to periodontal tissue to activate osteoclastogenesis and accelerate tooth movement in rats. The results showed that local RANKL gene transfer induced increased numbers of osteoclasts and accelerated tooth movement by approximately 150% in the rats in the experimental group compared to the control group. The effect of RANKL gene transfer was local and did not elicit any systemic effects. Interestingly, the number of osteoclasts was reduced time dependently after gene transfer [104]. Another group of investigators compared corticotomy with gene therapy using a hemagglutinating virus of Japan envelope vector containing mouse RANKL mRNA in rats for 32 days. The results showed increased level of RANKL protein 3 folds in the gene therapy group and 2 folds in the corticotomy group after 10 days; however, the level

regeneration, salivary gland disease and orthodontic treatment [102].

transplanted back into the patient [98–100].

Overall, the mechanism of tooth movement is complex and need strictly coordinated regulation of PDL, osteoclasts, osteocytes and osteoblasts. It is very challenging clinically to apply optimal force onto the tooth to avoid hyalinization. Clinically, tooth movement in patients is a result of combination of undermining and frontal resorption [83]. Compression sides involve increased expression of PGE2, TNF-α and IL-1β. PGE2 promotes osteoblast and osteoclast differentiation and activity. Activated osteoblasts secrete RANKL and OPG to trigger osteoclast differentiation and activity. TNF-α and IL-1β promote osteoclast differentiation and activity. In addition, matrix metalloproteinases (MMPs) expression is increased as well as the expression of M-CSF [84]. Loading compressive force affects osteocytes to upregulate the expression of connexin 43 [85], endothelial nitric oxide synthase (iNOS) [50], osteopontin [45], SOST [86] and RANKL [47]. These molecules recruit osteoclast precursors and activate osteoclasts to resorb the alveolar bone on the compression side. While on the tension side, increased expression of transforming growth factor-β (TGF-β), a potent ECM growth factor, was detected [87]. Several anabolic molecules such as bone sialoprotein (BSP) [88], collagen I (ColI) [89], vascular endothelial growth factor (VEGF) [84, 90], tissue inhibitors of metalloproteinases (TIMPs) [91], insulin-like growth factor (IGF) and its related receptor [92], heat shock protein 27 (HSP 27) [93] and ATP [94] were increasingly expressed on tension side during tooth movement. IL-6 around the osteocytes under loading can promote its signaling toward osteoblast pathway [53]. The presence of TIMPs around tension side is speculated to control the activity of MMP and remodeling pattern in alveolar bone. The anabolic events such as increased osteoblast activity and decreased osteoclast activity occur on the tension side of tooth movement.
