4. Studies on genetic manipulation of tooth movement

Administration of proteins that affect or activate osteoclasts could be a direct approach to modulate tooth movement though the dosage and side effects such as root resorption are factors of consideration. With modern advanced technology, the manufacturer can generate a large amount of human recombinant proteins for therapeutic purposes. However, the life span 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 transplanted back into the patient [98–100].

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

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

modulation of rate of tooth movement in animals [82].

58 Current Approaches in Orthodontics

activity occur on the tension side of tooth movement.

4. Studies on genetic manipulation of tooth movement

Administration of proteins that affect or activate osteoclasts could be a direct approach to modulate tooth movement though the dosage and side effects such as root resorption are factors of consideration. With modern advanced technology, the manufacturer can generate a large amount of human recombinant proteins for therapeutic purposes. However, the life span 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 regeneration, salivary gland disease and orthodontic treatment [102].

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 of RANKL protein was maintained in the gene therapy group but not in the corticotomy group. The number of osteoclasts in the RANKL gene therapy group was significantly higher at day 10 with or without tooth movement compared to the tooth movement only group. The tooth movement distance was 2 times more in the RANKL gene therapy group and 1.5 times in the corticotomy group; however, the rate of tooth movement slowed down in the corticotomy and controls groups but was constant in the RANKL gene therapy group. It was concluded that gene therapy was an alternative treatment for corticotomy to accelerate tooth movement and the efficacy of treatment was higher than corticotomy to accelerate tooth movement [106]. The OPG gene transfer experiment was performed by another group of investigators using the same viral envelope packaging and delivery system to investigate the inhibition of orthodontic relapse in rats. The first molars in the rats were moved mesially for 3 weeks then the springs were removed to generate orthodontic relapse in the rats. The rats received OPG gene therapy then were observed for 2 weeks. The results showed that relapse was significantly inhibited 2 times compared to the mock and control groups. The bone mineral density and bone volume fraction of alveolar bone were significantly increased in the gene therapy group compared to the mock and control groups. No difference of bone mineral density and bone volume fraction of tibia was found among groups. The investigators stated that local OPG gene therapy to periodontal tissues could inhibit relapse after orthodontic tooth movement via osteoclastogenesis inhibition [110]. The same group of investigators further investigated the effect of local OPG gene therapy on orthodontic root resorption with the same design of experiment. They utilized a microcomputed tomogram and histological analyses. The result showed no difference between root resorption at the beginning and the end of tooth movement in the OPG gene therapy group. However, the repair of root resorption in the gene therapy group was higher than other control groups [107]. Another study investigated the effect of local OPG gene therapy using mesenchymal stem cells as carriers for plasmid containing OPG mRNA. This cell mediated OPG gene transfer was generated by insertion of plasmid containing OPG mRNA into the mesenchymal stem cells and the cells were injected into the animals. The result showed that the cells containing OPG package grew in the animals' PDL and the number of osteoclasts, level of RANKL and bone resorption were reduced significantly after single injection. The level of OPG was highest in the gene therapy group [108].

targeting using endogenous microRNA (miRNA) has emerged as a powerful tool for targeted gene delivery. miRNAs are short, noncoding and highly conserved RNA sequences that tightly regulate the expression of genes by binding to their target sequence in the corresponding mRNAs [112, 113]. Majority of miRNA biogenesis involves transcription by RNA polymerase II to generate primary microRNA (pri-miRNA) followed by Drosha (RNase III enzyme) processing, which produces precursor miRNA (pre-miRNA). The pre-miRNA is transported to the cytoplasm via exportins/RanGTP complex. In the cytoplasm, the pre-miRNA is cleaved by another RNase III enzyme called Dicer to generate mature miRNA. The mature miRNA then forms a microRNA associated RNA-induced silencing complex (miRISC) with Argonaute proteins. The complex is steered to the target mRNA via base pairing with the target sequence of the miRNA. The degree of perfect complementarity at nucleotides 2–8 (binding sequence) in the 5<sup>0</sup>

Advances in Orthodontic Tooth Movement: Gene Therapy and Molecular Biology Aspect

http://dx.doi.org/10.5772/intechopen.80287

end of the miRNA is essential for a successful action of the RISC complex. Depending on the extent of complementarity with the target sequence, gene expression is repressed either by inhibition of translation or by cleavage of the corresponding mRNA [114]. The process of gene therapy using endogenous miRNAs involves selection process of miRNA candidates, design of expression cassettes if constant expression is needed, selection of delivery carrier, and evaluation of system in cells, animal models and clinical trials [114]. Several miRNAs have been reported for their expression and roles in PDL and alveolar bones [115–118]. Under loading, several miRNAs in PDL and alveolar bone respond to the loading force and orientation of forces in different pattern of expression [119–121]. miRNA-21 has been shown to have critical roles in PDL, osteoblasts and osteoclasts [120, 122–127]. In addition, miRNA-21 deficient mouse demonstrated delayed tooth movement compared to the control mice via inhibition of osteoclastogenesis [127]. miRNA-29 was reported as a crucial miRNA for alveolar bone remodeling during tooth movement due to its expression under different orientation of loading forces and its expression profile in crevicular fluid during tooth movement in human [121, 128]. miRNA-29 expression in human PDL was up-regulated under compression but down-regulated under stretch force orientation [121] and its expression on crevicular fluid increased along the course of tooth movement [128]. Moreover, miRNA-29 sponge transgenic mice demonstrated delayed tooth movement due to the decreased numbers of osteoclasts [129]. These microRNAs could be a target candidate for gene therapy for orthodontic tooth movement. There are viral and nonviral delivery systems in clinical trials for gene therapy. Among viral vector system, lentiviral vector-based system has been developed and tested for its safety for more than 10 years. Non-integrating lentiviral vector have been investigated as a means of avoiding insertional mutagenesis. However, there is a disadvantage of this approach regarding the short-lived of the vectors in dividing cells [130]. Nonviral gene delivery systems (nVGDS) have great potential for therapeutic purposes and have several advantages over viral delivery including lower immunogenicity and toxicity, better cell specificity, better modifiability, and higher productivity. However, there is no ideal nVGDS; hence, there is widespread research to improve their properties [97]. The nVGDS system includes chemicals, peptides, liposomes, and polymers [97]. Exosomes are small (30–150 nm in diameter) extracellular vesicles that are formed in multivesicular bodies and are released from cells as the multivesicular bodies fuse with the plasma membrane. The exosomes were proposed to be used for delivery of miRNAs, protein and oligonucleotide complex [131], and were found to be cell secreted from osteoclasts [131] and in gingival crevicular fluid during the course

of tooth movement [128].


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Gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections). This approach is still in the developing process as an alternative approach to treat deformity or disease that conventional method could not achieved. Although many clinical trials have shown the efficacy of the treatment, the technique remains risky and is still under processes of investigation to make sure that it will be safe and do not elicit any systemic or hereditary effects for the patients.
