H. Mitani

Effects of Topical Administration of a Bisphosphonate (Risedronate) on Orthodontic Tooth Movements in Rats

In orthodontics, undesirable movement of anchor teeth during tooth movement and relapse of moved teeth after treatment are the main causes of unsuccessful results. If these tooth movements could be prevented with pharmacological agents, a less complex orthodontic force system and less extensive retention would be required. The purpose of this study was to examine the effect of topical administration of a bisphosphonate (risedronate), a potent blocker of bone resorption, on orthodontic tooth movements in rats. In the first experiment, both the right and left upper first molars were moved buccally with a standardized expansion spring under administration of risedronate. Risedronate solution was injected into the sub-periosteum area adjacent to the left upper first molar. The right first molar served as a control with an injection of 0.9% NaCl solution. The topical administration of risedronate caused a significant and dose-dependent reduction of tooth movement after the orthodontic force was applied. In the second experiment, the right and left upper molars were first moved buccally for three weeks. The spring was then removed, and administration of risedronate was begun. The topical administration of risedronate inhibited relapse of the tooth in a dose-dependent manner. The administration of risedronate did not affect either overall growth of the animals or longitudinal growth of tibiae. These results suggest that topical application of risedronate may be helpful in anchoring and retaining teeth under orthodontic treatment.

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

SummaryImmunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.

Local RANKL gene transfer to the periodontal tissue accelerates orthodontic tooth movement

It has been reported that not only selective alveolar-bone resorption, but also receptor activator of nuclear factor kappa B ligand (RANKL) expression is induced on the compressed side of an orthodontically moving tooth. Numerous reports have described the pharmacological acceleration of tooth movement (TM) through the activation of osteoclasts. However, because of rapid flush out by blood circulation, daily systemic administration or daily local injection is needed. Previously, we discovered that every-3-days OPG gene transfer to the periodontal-tissue inhibited RANKL-mediated osteoclastogenesis and diminished experimental TM. Therefore, we hypothesized that local RANKL gene transfer into the periodontal tissue would accelerate TM. The upper first molars of 6-week-old male Wistar rats were moved palatally using fixed orthodontic wires. The inactivated hemagglutinating-virus of Japan (HVJ) envelope vector containing the mouse RANKL expression plasmid was injected periodically into the palatal periodontal tissue of the upper first molars during TM. Local RANKL gene transfer significantly enhanced RANKL expression and osteoclastogenesis in periodontal tissue without any systemic effects. The TM rate was significantly increased in the RANKL gene transfer side. In conclusion, we demonstrated that transfer of the RANKL gene to the periodontal-tissue activated osteoclastogenesis and accelerated the amount of experimental TM. Local RANKL gene transfer might be a useful tool not only for shortening orthodontic treatment, but also for moving ankylosed teeth where teeth, fuse to the surrounding bone.

Inhibitory Effect of the Topical Administration of a Bisphosphonate (Risedronate) on Root Resorption Incident to Orthodontic Tooth Movement in Rats

Root resorption associated with tooth movement is an unsolved problem in orthodontics. If such root resorption could be prevented, it would be an important contribution toward reducing risk factors in orthodontic treatment. The purpose of this study was to examine the effects of the topical administration of a bisphosphonate, risedronate, which is known to be a potent blocker of bone resorption, on root resorption during tooth movement and on the repair of the resorbed root surface after tooth movement in rats. In the first experiment, both the right and left upper first molars were moved buccally with a standardized expansion spring under administration of risedronate. After day 7, extensive root resorption had occurred on the control side, and the area of root resorption reached a maximum on day 14. The topical administration of risedronate caused a significant and dose-dependent inhibition of root resorption after the orthodontic force was applied. In the second experiment, the right and left upper molars were first moved buccally for 3 weeks. Risedronate treatment began on the day the spring was removed. After the force was withdrawn, the resorbed root surfaces on both the control and risedronate-treated sides were gradually restored by apposition of repair cementum (cementoid). The topical administration of risedronate did not appear to inhibit the repair process of root resorption. These results suggest that the topical administration of risedronate may be useful in preventing root resorption of teeth during orthodontic treatment.

Local OPG Gene Transfer to Periodontal Tissue Inhibits Orthodontic Tooth Movement

Previously, we discovered that RANKL expression is induced in compressed periodontal ligament cells, and that this promotes osteoclastogenesis on the compression side in orthodontic tooth movement. We hypothesized that local OPG gene transfer to the periodontium would neutralize the RANKL activity induced by mechanical compressive force, thereby inhibiting osteoclastogenesis and diminishing tooth movement. The upper first molars of six-week-old male Wistar rats were moved palatally by means of a fixed-orthodontic wire. A mouse OPG expression plasmid [pcDNA3.1(+)-mOPG] was constructed, and the production of functional OPG protein was confirmed in vitro. The inactivated HVJ envelope vector containing pcDNA3.1(+)-mOPG or PBS was injected periodically into the palatal periodontal tissue of upper first molars. When this local OPG gene transfer was performed, OPG production was induced, and osteoclastogenesis was inhibited. Local OPG gene transfer significantly diminished tooth movement. In this study, we report that OPG gene transfer to periodontal tissue inhibited RANKL-mediated osteoclastogenesis and inhibited experimental tooth movement.

Expression of MMP-8 and MMP-13 Genes in the Periodontal Ligament during Tooth Movement in Rats

Periodontal ligament tissue is remodeled on both the tension and compression sides of moving teeth during orthodontic tooth movement. The present study was designed to clarify the hypothesis that the expression of MMP-8 and MMP-13 mRNA is promoted during the remodeling of periodontal ligament tissue in orthodontic tooth movement. We used the in situ hybridization method and semi-quantitative reverse-transcription/polymerase chain-reaction analysis to elucidate the gene expression of MMP-8 and MMP-13 mRNA. Expression of MMP-8 and MMP-13 mRNA transiently increased on both the compression and tension sides during active tooth movement in vivo. The gene expression of MMP-8 and MMP-13 was induced by tension, while compression indirectly promoted the gene expression of MMP-8 and MMP-13 through soluble factors in vitro. Thus, we concluded that the expression of MMP-8 and MMP-13 is differentially regulated by tension and compression, and plays an important role in the remodeling of the periodontal ligament.

Effect of stretching on gene expression of β1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions

Differentiation of skeletal tissues, such as bone, ligament and cartilage, is regulated by complex interaction between genetic and epigenetic factors. In the present study, we attempted to elucidate the possible role of cell-extracellular matrix (ECM) adhesion on the inhibitory regulation in chondrogenesis responding to the tension force. The midpalatal suture cartilages in rats were expanded by orthopedic force. In situ hybridization for type I and II collagens, immunohistochemical analysis for fibronectin, alpha5 and beta1 integrins, paxillin, and vinculin, and cytochemical staining for actin were used to demonstrate the phenotypic change of chondrocytes. Immunohistochemical analysis for phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK)-1/2 was performed. The role of the cell-ECM adhesion in the response of the chondroprogenitor cells to mechanical stress and the regulation of gene expression of focal adhesion kinase (FAK) and integrins were analyzed by using an in vitro system. A fibrous suture tissue replaced the midpalatal suture cartilage by the expansive force application for 14 days. The active osteoblasts that line the surface of bone matrix in the newly formed suture tissue strongly expressed the type I collagen gene, whereas they did not express the type II collagen gene. Although the numbers of precartilaginous cells expressing alpha5 and beta1 integrin increased, the immunoreactivity of alpha5 integrin in each cell was maintained at the same level throughout the experimental period. During the early response of midpalatal suture cartilage cells to expansive stimulation, formation of stress fibers, reorganization of focal adhesion contacts immunoreactive to a vinculin-specific antibody, and phosphorylation and nuclear translocation of ERK-1/2 were observed. In vitro experiments were in agreement with the results from the in vivo study, i.e. the inhibited expression of type II collagen and upregulation in integrin expression. The arginine-glycine-aspartic acid-containing peptide completely rescued chondrogenesis from tension-mediated inhibition. Thus, we conclude that stretching activates gene expression of beta1 integrin and FAK and inhibits chondrogenesis through cell-ECM interactions of chondroprogenitor cells.

Cyclical Tensile Force on Periodontal Ligament Cells Inhibits Osteoclastogenesis through OPG Induction

The periodontal ligament (PDL) maintains homeostasis of periodontal tissue under mechanical tensile-loading caused by mastication. Occlusal load inhibits atrophic alveolar bone resorption. Previously, we discovered that continuous compressive force on PDL cells induced osteoclastogenesis-supporting activity, with up-regulation of RANKL. We hypothesized that, unlike compression, cyclical tensile force up-regulates OPG expression in PDL cells via TGF-beta up-regulation, and does not induce osteoclastogenesis-supporting activity. PDL cells were mechanically stimulated by cyclical tensile force in vitro. The conditioned media of PDL cells that had been subjected to cyclical tensile force inhibited osteoclastogenesis. Cyclical tensile force up-regulated not only RANKL mRNA expression, but also OPG mRNA expression in PDL cells. Tensile force up-regulated TGF-beta expression in PDL cells as well. Administration of neutralizing antibodies to TGF-beta inhibited OPG up-regulation under cyclical tensile-force stimulation in a dose-dependent manner. Additionally, the osteoclastogenesis-inhibitory effect of the conditioned media of PDL cells under cyclical tensile force was partially rescued by the administration of TGF-beta neutralizing antibodies. In conclusion, tensile force inhibited the osteoclastogenesis-supporting activity of PDL cells by inducing the up-regulation of OPG via TGF-beta stimulation.

Effects of bisphosphonates on alkaline phosphatase activity, mineralization, and prostaglandin E2 synthesis in the clonal osteoblast- like cell line MC3T3-E1

The effects of 3 bisphosphonates, AHBuBP, AHPrBP, and Cl2MBP on cell growth, alkaline phosphatase (ALP) activity, mineralization, and prostaglandin E2 (PGE2) synthesis in the clonal osteoblast-like cell line MC3T3-E1 were studied. These bisphosphonates had essentially similar effects on growth and the osteoblastic functions of the cells, i.e., they had no inhibitory effects on cell growth except at higher concentrations, they increased ALP activity, and inhibited PGE2 production. In the presence of AHBuBP, ALP activity was higher than that in the control after day 6 of culture. Lower concentrations of AHBuBP slightly facilitated mineralization by the cells. It is probable that bisphosphonates enhance the functions of osteoblasts in certain concentration and that the inhibition of endogenous PGE2 production may be involved in the mechanism of action of bisphosphonates.

Effects of expansive force on the differentiation of midpalatal suture cartilage in rats

In an attempt to clarify the effects of biomechanical tensional force on chondrogenic and osteogenic differentiation of secondary cartilage, the midpalatal sutures of 4-week-old Wistar male rats were expanded by orthodontic wires which applied 20 g force for 4, 7, 10, and 14 days. The differentiation pathways in the midpalatal suture cartilage were examined by immunohistochemistry for osteocalcin, type I and type II collagen, and von Kossa histochemistry. Although the midpalatal sutures of the control animals consisted mainly of two separate secondary cartilages with mesenchyme-like cells at their midlines, type I collagen-rich fibrous tissue began to appear at day 4 and increased at the midline of the cartilage with days of experiment. At the end of the experiment, type I collagen-rich and calcified bone matrix appeared at the boundary between the precartilaginous and the cartilaginous cell layers. Most of the cartilaginous tissues were separated from each other and the midpalatal suture was replaced by osteocalcin-positive intramembranous bone and fibrous sutural tissue. These results strongly suggest that tensional force changed the phenotypic expression of collagenous components in secondary cartilage, which may reflect the differentiation pathway of osteochondro progenitor cells.