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Objective: Regulation of alveolar bone metabolism is required in clinical dentistry. The aim of the present study was to establish a method for gene transfer into the periodontal ligament (PDL) by in vivo electroporation with a plasmid vector and to investigate the effects of BMP-4 transfer into the PDL., Design: Plasmids containing mouse BMP-4 cDNA (pCAGGS-BMP4) were transfected into cultured rat PDL cells by in vitro electroporation, and BMP-4 production and secretion were detected by immunocytochemistry and western blotting. Next, pCAGGS-BMP4 was injected into the PDL of rats, and electroporation was performed in vivo, using original paired-needle electrodes. BMP-4 expression was examined by immunohistochemical staining 3, 7, 14, 21, and 28days after electroporation. Control groups were injected with pCAGGS by electroporation, injected with pCAGGS-BMP4 without electroporation, or subjected to neither injection nor electroporation., Results: In vitro-transfected rat PDL cells exhibited production and secretion of the mature-form BMP-4. After in vivo electroporation of pCAGGS-BMP4, site-specific BMP-4 expression peaked on day 3, gradually decreased until day 14, and was absent by day 21. We observed no unfavorable effects such as inflammation, degeneration, or necrosis., Conclusions: Gene transfer by electroporation with plasmid DNA vectors has several advantages over other methods, including the non-viral vector, non-immunogenic effects, site-specific expression, simplicity, cost-effectiveness, and limited histological side effects. Our results indicate that the method is useful for gene therapy targeting the periodontal tissue, which regulates alveolar bone remodeling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

During orthodontic tooth movement, the activation of the vascular system in the compressed periodontal ligament (PDL) is an indispensable process in tissue remodeling. We hypothesized that compressive force would induce angiogenesis of PDL through the production of vascular endothelial growth factor (VEGF). We examined the localization of VEGF in rat periodontal tissues during experimental tooth movement in vivo, and the effects of continuous compressive force on VEGF production and angiogenic activity in human PDL cells in vitro. PDL cells adjacent to hyalinized tissue and alveolar bone on the compressive side showed marked VEGF immunoreactivity. VEGF mRNA expression and production in PDL cells increased, and conditioned medium stimulated tube formation. These results indicate that continuous compressive force enhances VEGF production and angiogenic activity in PDL cells, which may contribute to periodontal remodeling, including angiogenesis, during orthodontic tooth movement.

Introduction: Accelerating the speed of orthodontic tooth movement should contribute to the shortening of the treatment period. This would be beneficial because long treatment times are a negative aspect of orthodontic treatment. In this study, we evaluated the effects of mechanical stimulation by resonance vibration on tooth movement, and we showed the cellular and molecular mechanisms of periodontal ligament responses., Methods: The maxillary first molars of 6-week-old male Wistar rats were moved to the buccal side by using an expansive spring for 21 days (n = 6, control group), and the amount of tooth movement was measured. Additional vibrational stimulation (60 Hz, 1.0 m/s(2)) was applied to the first molars by using a loading vibration system for 8 minutes on days 0, 7, and 14 during orthodontic tooth movement (n = 6, experimental group). The animals were killed under anesthesia, and each maxilla was dissected. The specimens were fixed, decalcified, and embedded in paraffin. Sections were used for immunohistochemical analysis of receptor activator of NF kappa B ligand (RANKL) expression. The number of osteoclasts in the alveolar bone was counted by using TRAP staining, and the amount of root resorption was measured in sections stained with hematoxylin and eosin., Results: The average resonance frequency of the maxillary first molar was 61.02 +/- 8.38 Hz. Tooth movement in the experimental group was significantly greater than in the control group (P <.05). Enhanced RANKL expression was observed at fibroblasts and osteoclasts in the periodontal ligament of the experimental group on day 3. The number of osteoclasts in the experimental group was significantly increased over the control group on day 8 (P <.05). Histologically, there were no pathological findings in either group or significant differences in the amount of root resorption between the 2 groups., Conclusions: The application of resonance vibration might accelerate orthodontic tooth movement via enhanced RANKL expression in the periodontal ligament without additional damage to periodontal tissues such as root resorption.

Periodontal ligament (PDL) cells play an essential role in orthodontic tooth movement. We recently reported that clodronate, a non-N-containing bisphosphonate, strongly inhibited tooth movement in rats, and thus could be a useful adjunct for orthodontic treatment. However, it is not clear how clodronate affects the responses of PDL cells to orthodontic force. In this study, we hypothesized that clodronate prevents the mechanical stress-induced production of prostaglandin E(2) (PGE(2)), interleukin-1beta (IL-1beta), and nitric oxide (NO) in human PDL cells. A compressive stimulus caused a striking increase in PGE(2) production, while the responses of IL-1beta and NO were less marked. Clodronate concentration-dependently inhibited the stress-induced production of PGE(2). Clodronate also strongly inhibited stress-induced gene expression for COX-2 and RANKL. These results suggest that the inhibitory effects of clodronate on tooth movement and osteoclasts may be due, at least in part, to the inhibition of COX-2-dependent PGE(2) production and RANKL expression in PDL cells.

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.

Objective: We examined the effect of the pretreatment of roots with alendronate on the restoration of the support function of the healing periodontal ligament in replanted rat molars., Methods: The left maxillary first molars were extracted, placed in 0.9% NaCl containing 1 mm alendronate (alendronate group) or 0.9% NaCl (control group) for 5 min, and were replanted into their sockets. Groups of animals were killed at 7, 14, and 21 days after replantation. Normal control rats were also killed on the same days. The force required to extract the replanted or normal tooth from its socket was measured, and a load-deformation curve was developed and analyzed. Micro-computed tomography and histologic analyses were also made., Results: The mechanical properties of the healing periodontal ligament in the alendronate group were gradually restored from 7 to 21 days. However, fractures of the roots and bones during mechanical testing occurred in most of the replanted teeth in the control group at 21 days. The rates of restoration of the mechanical strength, extensibility, stiffness, and toughness for the alendronate group at 21 days were 67, 98, 74, and 68% of the normal controls, respectively. Micro-computed tomography and histologic observations revealed that bone-like structures within the pulp and ankylosis between the roots and socket bones occurred commonly in the control group, but were uncommon in the alendronate group., Conclusions: Our findings suggest that the pretreatment with alendronate inhibits the formation of abnormal mineralized tissues and results in better restoration of the support function of the healing periodontal ligament in replanted teeth., ((c)Blackwell Munksgaard 2004)

Objective: We examined the stress-strain and stress-relaxation properties of the periodontal ligament (PDL) in the rat molar at 2, 6, 12, and 24 months of age to elucidate age-related changes in the tooth support function of the PDL., Design: From the dissected left and right mandibles in each rat, a pair of transverse sections (ca. 0.45 mm in thickness) of the first molar was cut at the middle part of the mesial root. We then obtained a load-deformation curve for the PDL, using one of the paired sections. The other section was loaded to as much as 50% of the maximum load as determined from the contralateral section, and keeping the deformation constant for 10 min, a load-relaxation curve was obtained and analysed., Results: The maximum shear stress and tangent modulus decreased between 2 and 24 months of age. As the maximum shear strain increased with age (P < 0.001), the failure strain energy density did not change between 2 and 24 months of age. The stress-relaxation during the 10 min period decreased from 2 to 24 months of age (P < 0.01). The relaxation process of the PDL in each age was well described by a sum of three exponential decay functions. The age-related decrease in the relaxation was found to be mainly due to the increase in the relaxation time for the long-term relaxation component., Conclusion: These results indicate that the maximum shear stress and stiffness of the rat molar PDL decrease between 2 and 24 months of age; but its toughness remains unchanged due to an increase in the extensibility. Our findings further indicate that the fluid flow and movements of macromolecules within the stretched PDL fibres during the stress-relaxation decrease with advancing age.

Groups of rats were killed at 2, 6, 12, and 24 months of age. From dissected left and right mandibles in each rat, three pairs of transverse sections were cut at the incisal, middle, and basal regions of the incisor. One section in each pair was loaded until failure and a stress-strain curve for the periodontal ligament (PDL) was obtained. The other section was loaded to up to 50% of the maximum shear stress as determined from the contralateral section and then kept at a constant strain for 10 min, to obtain the stress-relaxation curve at the same region of the PDL. The maximum shear stress and toughness increased with age at the incisal region and the maximum shear strain increased with age at the incisal and middle regions. The tangent modulus decreased with advancing age at the middle region. The stress-relaxation during 10 min decreased with advancing age at the incisal and basal regions, but not at the middle region. The relaxation process was well described by a sum of three exponential decay functions, reflecting the short-, medium-, and long-term relaxation components. The age-related decrease in the relaxation was mainly attributable to increases in the ratio and relaxation time of the long-term relaxation component. These results suggest that with advancing age the mechanical strength and toughness of the PDL are enhanced mostly at the incisal region and that the viscous fraction is relatively decreased at the incisal and basal regions along the long axis of the rat incisor.

The periodontal ligament (PDL) is a specialized, mechanically responsive tissue that adapts via cellular responses to equilibrate the effects of mechanical stress on teeth. However, the mechanism of remodelling by which individual cells in periodontal tissue detect and respond to mechanical stress is not well understood. To identify the cellular mechanisms induced by mechanical stress in the periodontal ligament, we examined the effects of cyclic stretching on periodontal ligament fibroblast-like cells (PDL cells). Furthermore, we investigated the effects of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and interaction with peripheral blood mononuclear cells (PBMCs) on mechanically-simulated PDL cells. PDL cells were cultured on type I collagen-coated silicon membranes with 10% FBS alpha-MEM, and then subjected to cyclic mechanical stimulation (1 s stretching/1 s relaxation, 15% maximum elongation). Alkaline phosphatase activity was monitored by cytochemical and spectrophotometric methods. Morphologically, the cells assumed a spindle shape, and the cytoskeletal components, including microtubules and F-actin filaments, were aligned perpendicular to the strain force vector. Cyclic stretching decreased ALPase activity in PDL cells. The anabolic systemic hormone 1,25(OH)(2)D(3) increased ALPase activity, but this effect was suppressed by cyclic stretching. ALPase activities were reduced by co-culture with PBMCs, including lymphocytes and monocytes. This PBMC-induced ALPase reduction was synergistically reduced by cyclic stretching. ALPase activity was decreased by co-culture with PBMCs, and ALPase activity was reduced synergistically by treatment with PBMCs and cyclic stretching. We conclude that PDL cells changed their shape and alignment in response to cyclic stretching. Furthermore, local factors, such as mechanical stress and PBMCs, showed synergistic suppressive effects on ALPase activity., (Copyright 2004 John Wiley & Sons, Ltd.)

Biomechanical properties and morphological features of the periodontal ligament (PDL) in the rat mandibular molars were examined during orthodontic retention. Seventy-three male rats of the Wistar strain, 8 weeks of age, were used for biomechanical analysis and six rats for morphological analysis. An elastic band was inserted between the mandibular first and second molars for 4 days; after removal of the elastic band the interdental space was filled with resin for 4 and 8 days. The maximum shear stress, tangent modulus, and failure strain energy density of the PDL of the first molar in the experimental animals decreased markedly following application of an orthodontic force. They increased rapidly and were restored completely to the control levels by the 8th day after retention. Light microscopy showed severe compression and extension of the PDL in the experimental animals on the 8th day after retention. Birefringent collagen fibre bundles running across the compressed and expanded PDL were observed, although they appeared to be thinner with less insertions into the alveolar bone or cementum in the experimental animals than in the controls. This suggests that the periodontal collagen fibres were partially reorganized and rearranged during retention. The reorganization and rearrangement of periodontal collagen fibres seemed to be partly related to the restoration of mechanical strength of the rat molar PDL during the 8 days of retention.

We prepared decalcified sagittal sections (20 microm thick) from the incisal, middle, and basal regions of the mandibular incisor of male Wistar rats aged 2, 6, 12, and 24 months, and examined the sections using polarized light microscopy. Most of the birefringent fibers appeared to run obliquely across the periodontal ligament. Birefringent fibers running parallel to the long axis of the incisor were also found in the intermediate area of the ligament. Similar fiber architecture was observed in all four age groups. Quantitative analysis showed that the retardation values of collagen were higher in the bone- and tooth-related areas and lower in the intermediate area of the ligament. The values for the bone- and tooth-related areas increased from the basal toward the incisal regions in all four age groups. Age-related changes in the retardation values were found only in the incisal region of the incisor. In the incisal region, the values for the bone- and tooth-related areas increased markedly from 2-24 months of age, whereas those for the intermediate area increased slightly but significantly with age. Our findings indicate that the degrees of molecular organization and alignment of collagen fibers in the bone- and tooth-related areas of the ligament are higher than those in the intermediate area and increase near the incisal region and with age. It is also suggested that the collagen fibers in the intermediate area remain immature along the long axis of the incisor throughout the life span of the animal., (Copyright 2002 Wiley-Liss, Inc.)

The periodontal ligament is subjected to mechanical loading during occlusion and mastication. Although internuclear transcription factors are associated with the regulatory pathway that converts these extracellular mechanical stimuli into a cellular response, there are no reports on these in human periodontal ligament fibroblasts. In this study, the amounts of c-fos mRNA in human periodontal ligament fibroblasts were investigated shortly after subjecting them to a cyclic tension force in vitro. The mRNA of alkaline phosphatase and the matrix proteins type I collagen, type III collagen, matrix Gla-protein, osteonectin, osteopontin, and osteocalcin were also examined. A significant, rapid, transient increase in c-fos mRNA was detected, which peaked 30 min after the application of mechanical force. However, there was no significant change in the mRNA for alkaline phosphatase or the matrix proteins. These results provide evidence that periodontal ligament fibroblasts and c-fos may play a critical part in the response of periodontal tissue to mechanical stimulation.

Previously, we discovered that periodontal ligament (PDL) cells not only support osteoclastogenesis through cell-to-cell contact, but also inhibit the formation of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells by a producing soluble factor(s). Furthermore, PDL cells express both receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) messenger RNA (mRNA). Clinically, "ankylosed teeth," which lack periodontal ligament, cannot be moved with orthodontic tooth treatment. From this, we hypothesized that PDL cells under mechanical stress should play a pivotal role in osteoclast formation during orthodontic tooth movement. This study examined how mechanical stress affects the osteoclastogenesis-supporting activity of PDL cells. PDL cells were compressed continuously and then cocultured with peripheral blood mononuclear cells (PBMCs) for 4 weeks. PDL cells under mechanical stress up-regulated osteoclastogenesis from PBMCs. Furthermore, the expression of RANKL mRNA and protein in PDL cells increased with compressive force in parallel with the change in the number of osteoclasts. In addition, cyclo-oxygenase 2 (COX-2) mRNA expression was induced by compressive force, and indomethacin inhibited the RANKL up-regulation resulting from compressive force. PDL cells under compressive force exhibited significantly increased prostaglandin E2 (PGE2) production in comparison with control PDL cells. Exogenous PGE2 treatment increased RANKL mRNA expression in PDL cells. Interestingly, OPG expression remained constant throughout compressive force or PGE2 treatment. In conclusion, compressive force up-regulated RANKL expression in PDL cells. Furthermore, RANKL up-regulation in mechanically stressed PDL cells was dependent on PGE2.

Three groups of male Wistar rats received daily subcutaneous injections of 10 mg/kg of hydrocortisone (HC group), 300 mg/kg of beta-aminopropionitrile (BAPN group), or saline (control group), for 10 days. The shear stress-strain and stress-relaxation properties of the incisor periodontal ligament were examined in transverse sections from dissected mandibles. Both the maximum shear stress and failure strain energy density increased significantly following the administration of hydrocortisone. The maximum shear stress decreased following the administration of BAPN. However, the stress-relaxations in the initial 10 min did not show significant differences among the three groups. Polarized light microscopic analysis revealed that the retardation value of the collagen fibers was highest in the HC group and lowest in the BAPN group for the bone-related area, but not for the tooth-related and middle areas of the ligament. It is suggested that the changes induced by hydrocortisone or BAPN occurred mainly in the elastic components and to a minor extent in the viscous components although the physical and biomechanical properties are determined by the interaction of all the various components. We also suggest that the main response to the drugs occurred in the collagen fibers in the bone-related area of the ligament.

We analyzed stress-strain curves in transverse sections at three different root levels of the cervical, middle, and apical regions of the distal root of the mandibular first molar in hamsters fed a high-carbohydrate (experimental group) or standard (control group) diet for 8 weeks. Each section was loaded in vitro at a rate of 5 mm/min in an extrusive direction. The rupture sites and the structures of birefringent collagen fibers in the periodontal ligament were also analyzed. The maximum shear stress, tangent modulus, and failure strain energy density of the periodontal ligament were significantly greater in the cervical region than in the middle and apical regions in both the control and experimental groups. The maximum shear stress, tangent modulus, and failure strain energy density of the periodontal ligament in the experimental group were significantly lower than those in the control group at the cervical and middle regions. The maximum shear strain was significantly greater in the experimental group than in the control group at the middle and apical regions. Histological sections showed that the periodontal ligament ruptured irregularly in both groups. The collagenous fibers of the ligament in the experimental group appeared to be thinner and less birefringent at the cervical and middle regions of the root. These results suggest that a high-carbohydrate diet differentially affects the collagen fibers in the periodontal ligament as a function of the root level, and that these effects are evident in changes in the mechanical properties of the ligament.

Tooth-periodontal ligament-bone segments were cut in the form of rectangular prisms (1.5 mm wide, 0.65 mm thick, and long enough to allow anchorage of the bone and tooth-end portions in a stretching jig) from the mandibular incisors of 10 rabbits. The experimental set-up enabled simultaneous recording, on video, of the changing image brightness under polarizing optics together with extension across the periodontal ligament. Specimens were stretched until failure at a velocity of 0.5 mm/min. The tensile load-deformation curve of the ligament exhibited an initial, non-linear region that was followed by a linear region, a subsequent yielding region preceding the maximum point, and a final descending region. Gradual increases in the intensity of birefringence in the linear and yielding regions indicated that stress concentrations occur in the supporting fibres attached to mineralized tissues. In the final descending region of the curve, progressive breakages of individual fibre bundles occurred, mainly in the middle zone of the ligament. Analysis of the polarized light-microscopic images showed that the increases in brightness and area of birefringent collagen fibre bundles occurred in parallel with the stress generated. These results suggest that the collagen fibre bundles became aligned with the direction of loading and the intensity of their birefringence increased according to the applied tensile force.

Periodontal ligament (PDL) cells play an important role in maintaining the homeostasis of periodontal tissues. However, it is not known how PDL cells contribute to osteoclastogenesis. In this study, we examined the consequences of cell-to-cell interactions between peripheral blood mononuclear cells (PBMCs) and PDL cells during osteoclastogenesis. PBMCs were co-cultured directly or indirectly with PDL cells for two to four weeks. PBMCs that were directly co-cultured with PDL cells formed significantly more resorption pits on dentin slices than did PBMCs that were cultured alone. However, soluble factor(s) produced from PDL cells inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Furthermore, PDL cells expressed both receptor activator nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) mRNA. In conclusion, PDL cells support osteoclastogenesis through cell-to-cell contact. PDL cells might regulate osteoclastogenesis by opposing mechanisms--stimulation of resorptive activity by RANKL and inhibition by OPG--thus affecting processes such as periodontitis and orthodontic tooth movement.

Background: The aim of this study was to obtain a more precise understanding of the mechanical properties of the periodontal ligament in continuously erupting incisors by comparing the shear stress-strain relations among teeth from four closely related species., Methods: Four species of experimental animals (mice, hamsters, rats, and rabbits) were used. Transverse sections of the left mandibular incisors were cut from the incisal, middle, and basal regions of each incisor. The tooth was pushed out of the alveolar bone in an extrusive direction at 5 mm/min using a materials testing machine. The maximum shear stress, maximum shear strain, tangent modulus, and failure strain energy density were estimated from the resulting stress-strain curve. Polarized light microscopic observations of collagen fibers were also made., Results: All the biomechanical measures tended to decrease from the incisal toward the basal regions in all species. There were large species differences, especially in the incisal region, with the greatest maximum shear stress and failure strain energy density in hamsters. The greatest tangent modulus and the smallest maximum shear strain were observed in mice. The birefringent fiber architectures of the periodontal ligaments in the four species appeared to be similarly organized; the incisal periodontal ligament appeared to have more organized and thicker collagen fibres than did the middle and basal ligaments in the four species., Conclusions: These results suggest that the regional differences in the biomechanical properties of the periodontal ligament depend upon the developmental stages of the periodontal collagen fibers that may be related to the general arrangement, diameters, and densities of the collagen fiber bundles and the fiber insertions into the alveolar bone and cementum. The species differences in the biomechanical properties may be due to differences in the width of the periodontal ligament and the waviness as well as the strength and stiffness of the periodontal collagen fibers.

Stress-strain relations of this ligament in transverse sections of the mandibular first molar were examined over a wide range of velocities of loading from 1 to 10(4) mm/24 h. With increasing velocities, the maximum shear stress, tangent modulus and failure strain-energy density increased but the maximum shear strain decreased. The mechanical responses at the highest velocity for the molar ligament were compared with those previously found for the incisor ligament. Mechanical strength, stiffness and toughness were greater for the molar than for the incisor ligament; the molar ligament therefore has more extensible fibres or a different fibre arrangement. Comparison of the mechanical responses at the slowest velocity suggests that, though the stress level was greatly reduced (presumably because of stress relaxation), the fibre components of the molar ligament still reacted at this velocity. It is also suggested that the differences in the ratios of the mechanical measures in 10(4)-1 mm/24 h between the two types of teeth are due partly to their different periodontal fibre architectures.

Stress-strain curves of the periodontal ligament (PDL) were obtained at various velocities of extrusive loading of 1, 10, 10(2), 10(3) and 10(4) mm/24 h in vitro. Significant increases of the maximum shear stress, tangent modulus and failure strain energy density were found with increases in the velocity of loading. The maximum shear strain increased from a velocity of 1 to 10 mm/24 h but decreased from 10 to 10(4) mm/24 h. It was shown histologically that the free surface of the PDL adhering to the cementum after mechanical testing was rough and irregular at higher velocities and rather smooth at lower velocities. These results showed that the mechanical properties and mode of failure of the rat incisor PDL were greatly dependent on the strain rate. It is possible that the PDL of the continuously erupting rat incisor has mechanical characteristics favourable for resisting weakly to slow and small eruptive forces but strongly to the fast and large occlusal forces as suggested previously [Chiba and Komatsu, The Biological Mechanisms of Tooth Eruption and Root Resorption (1988)].

The load-deformation curves obtained by extraction of the rat maxillary first molar from its socket in the dissected jaw were analyzed so that the effect of orthodontic retention on the mechanical properties of the periodontal ligament could be examined. An elastic band was inserted between the rat maxillary first and second molars for four days, and then the interdental space was filled with resin for four or eight days. The average interdental spaces between the teeth ranged from 315 to 398 microns during the experimental period. The maximum shear load, elastic stiffness, and failure energy in shear decreased markedly following application of an orthodontic force, but they increased gradually and reached control levels on the 8th day after the retention. Maximum shear deformation at maximum load was not significantly different between the experimental and control teeth during the experimental period. It is suggested that, following orthodontic tooth movement, occlusal function was restored after a relatively short retention period, as was the impaired mechanical strength of the periodontal ligament.

The ultimate loads required to extract three mandibular molars in the dissected jaw were examined after elimination of the antagonistic teeth for up to 64 days. The ultimate loads in the experimental first and second molars decreased rapidly in the first few days, while those in the third molar remained low during the same period in both experimental and control groups. The ultimate loads in all three molars of the experimental animals then increased gradually towards the end of the experiment at rates similar to those in controls. Maximum relative reductions of the ultimate load were observed within the first 8 days in all three molars. An increase in the length of roots was also found in all hypofunctional molars. Daily rates of root elongation ranged from 13 to 19 microns/day in the control and from 18 to 26 microns/day in the experimental molars during the whole experiment. The greatest value (80 microns/day) was obtained during the first 8 days in the third molars of both control and experimental animals. Thus the mechanical strength of the periodontal ligament estimated in vitro may be increased by the development of teeth and by non-functional occlusal contacts with the opposing gingiva-covered alveolar ridge deprived of its tooth crowns. The ligament of the third molar was apparently immature at the beginning of the experiment.

Mechanical properties of the periodontal ligament of the incisor have been examined in sections of the mandibles of rats from 6 to 24 months of age. Mechanical measures estimated from the load-deformation and stress-strain curves did not show age-related changes, although the body and mandible weights increased gradually during the experimental period. It is suggested that qualitative and quantitative changes in the supporting tissues/unit area of the ligament are minor at a restricted region examined.

Age-related changes in the mechanical strength of the periodontal ligament have been examined by measuring the force required to extract the mandibular first molar from its socket at an extension rate of 5 mm/min in the dissected jaws of growing young rats from 3 to 32 weeks of age. Almost linear increase in the ultimate loads was found from 3 to 8 weeks of age. However the values from 8 to 32 weeks of age did not show any significant differences. Close correlations were found between the ultimate loads and the weights of animals, mandibles and extracted teeth and between the load and length of the tooth from 3 to 8 weeks of age. It is supposed that a mechanical equilibrium has been established within the periodontal ligament in association with various external and internal factors such as root elongation, alveolar bone growth, turnover and maturation of periodontal ligament and occlusal activity, at about 8 weeks of age in the rat mandibular first molar.

Mechanical testing was performed in various regions of the ligament at a velocity of intrusive loading of 5 mm/min. The perimeters of the lingual cementum and socket wall and the sectional area of the ligament were measured radiographically. Load-deformation curves were transformed into stress-strain curves using the area and width of the lingual part of the ligament. The mechanical properties of the ligament differed markedly along the long axis of the incisor; mechanical measures from the load-deformation and stress-strain curves decreased gradually from the incisal towards the basal regions. It is suggested that these regional differences in mechanical properties were much more marked than those in teeth of limited growth or in other connective tissues, and that the nature of the collagen fibres, together with surrounding cells, ground substance and blood vessels, may be important in determining such differences.

Effects of lathyrogens such as aminoacetonitrile (AAN), beta-aminopropionitrile (BAPN) and cysteamine--known inhibitors of cross-linking of collagen--on the mechanical strength of the periodontal ligament of the rat mandibular first molar were examined by measuring the ultimate load required to extract the tooth from its socket in the dissected jaw. Single injections of AAN (40 approximately 100 mg/100 g body weight) or of BAPN (100 mg/100 g body weight) caused significant decreases of the mechanical strength 24 h after administration of the drugs but that of cysteamine (30 mg/100 g body weight) did not. Significant correlations between the dose of AAN or of BAPN and the mechanical strength were found following daily administrations of the drugs for 5 days. The relative potency of AAN to BAPN was estimated to be 4.5 by a slope ratio assay. The rapid appearance and disappearance of the effect of lathyrogens on the mechanical strength of the periodontal ligament of the rat mandibular first molar provide further evidence that the turnover of the collagen in the tissue is fast. The half-time of collagen synthesis was estimated to be approximately 3 d.

The right maxillary molars of male rats were removed under ether anaesthesia to eliminate occlusal contact with the mandibular molars. Groups of rats were killed at 1, 2, 4, 8 and 16 days after the experimental procedure. The dissected mandibles were radiographed and length of erupted portion of the tooth, height of alveolar crest and length of tooth were measured. The tensile strength of the periodontal ligament was measured by extracting the first molar from its socket. A marked and progressive decrease of the load required to extract the tooth was found in the first few days after the removal of the opposing teeth. No significant difference in extracting loads was found between the 8- and 16-day groups. Radiography showed that the teeth had erupted, that the height of alveolar crest had decreased and that the length of the root had increased during the experimental period. These changes were detectable only on the 8th or 16th day following the experimental procedure. It is suggested that the reduction in the mechanical strength of the hypofunctional rat molar periodontal ligament is closely associated with the progressive atrophy of the periodontal ligament and that normal functional activity of the teeth is important not only for maintenance of the structural integrity of the periodontal ligament but also to maintain the mechanical strength of the supporting tissues.

Periodontal ligament (PDL) cells play an essential role in orthodontic tooth movement. We recently reported that clodronate, a non-N-containing bisphosphonate, strongly inhibited tooth movement in rats, and thus could be a useful adjunct for orthodontic treatment. However, it is not clear how clodronate affects the responses of PDL cells to orthodontic force. In this study, we hypothesized that clodronate prevents the mechanical stress-induced production of prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and nitric oxide (NO) in human PDL cells. A compressive stimulus caused a striking increase in PGE2 production, while the responses of IL-1β and NO were less marked. Clodronate concentration-dependently inhibited the stressinduced production of PGE2. Clodronate also strongly inhibited stress-induced gene expression for COX-2 and RANKL. These results suggest that the inhibitory effects of clodronate on tooth movement and osteoclasts may be due, at least in part, to the inhibition of COX-2-dependent PGE2 production and RANKL expression in PDL cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Three groups of male Wistar rats received daily subcutaneous injections of 10 mg/kg of hydrocortisone (HC group), 300 mg/kg of β-aminopropionitrile (BAPN group), or saline (control group), for 10 days. The shear stress-strain and stress-relaxation properties of the incisor periodontal ligament were examined in transverse sections from dissected mandibles. Both the maximum shear stress and failure strain energy density increased significantly following the administration of hydrocortisone. The maximum shear stress decreased following the administration of BAPN. However, the stress-relaxations in the initial 10 min did not show significant differences among the three groups. Polarized light microscopic analysis revealed that the retardation value of the collagen fibers was highest in the HC group and lowest in the BAPN group for the bone-related area, but not for the tooth-related and middle areas of the ligament. It is suggested that the changes induced by hydrocortisone or BAPN occurred mainly in the elastic components and to a minor extent in the viscous components although the physical and biomechanical properties are determined by the interaction of all the various components. We also suggest that the main response to the drugs occurred in the collagen fibers in the bone-related area of the ligament. [ABSTRACT FROM AUTHOR]