Chihiro Arai

Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells.

Among the various kinds of fibroblasts existing in the human body, the periodontal ligament (PDL) fibroblasts have been suggested as multipotent cells. Periodontal ligament fibroblasts are characterized by rapid turnover, a high remodeling capacity and remarkable capacity for renewal and repair. They also differentiate into osteoblasts and cementoblasts. We established iPS cells from human PDL fibroblasts by introducing the ES cell markers Oct3/4, Sox2, Nanog, Klf4 and Lin28 by retrovirus transduction, even without the oncogene c-Myc. The iPS cells established in this study expressed the ES cell markers and formed teratomas in SCID mice. The c-Myc expression level in the PDL fibroblasts was higher than that in the iPS cells by quantitative RT-PCR. Therefore, we have concluded that PDL fibroblasts could be an optimal cell source for iPS cells.

Time-lapse observation of rat periodontal ligament during function and tooth movement, using microcomputed tomography

The aim of this study was to observe the time-lapse changes in the rat periodontal ligament (PDL) during function and tooth movement. Under Nembutal anaesthesia, time-lapse changes in the thickness of the PDL of the first molars were investigated in five 12-week-old adolescent rats with microcomputed tomography. Three-dimensional (3D) images were reconstructed from the data. Histological observation was also performed, using undecalcified frozen sections of the maxillary first molar area. The PDL appeared as a radiolucent furrow on the 3D images. A slight change in the thickness of the PDL was observed 1 hour after initiation of orthodontic force loading, which became significant after 6 hours, with the appearance of pressure-tension zones during the tooth movement. These changes were more significant 3 days after orthodontic loading. Histological observation of the lingual cervical PDL (pressure zone) in nine 12- to 13-week-old rats demonstrated that the periodontal space had become narrow and the cellular elements appeared to be densely packed in the narrowed PDL 6 hours after orthodontic loading. Degeneration of tissues appeared 3 days after loading. Observation of the buccal cervical PDL (tension zone) demonstrated that the PDL was extended 6 hours after orthodontic force loading, and the extension continued for up to 3 days. Alkaline phosphatase activity was distributed in the PDL, except for the degenerating tissues in the pressure zone 3 days after loading. The results suggest that the periodontal reaction was initiated within 6 hours after orthodontic force loading, which was related to the structural changes of the PDL. The changes probably induced an early response in individual cells of the PDL.

Immunocompetent cells and cytokine expression in the rat periodontal ligament at the initial stage of orthodontic tooth movement

OBJECTIVE The aim of the present study is to investigate the involvement of immunocompetent cells, pro-inflammatory cytokines and HSP, to evaluate a change of periodontal ligament during the initial stage of orthodontic tooth movement. DESIGN In the present study, we investigated the distributional density of immunocompetent cells, the localisation of cytokines, and the expression levels of their mRNA in the periodontal ligament during the initial stage of orthodontic tooth movement, using immunohistochemistry and real-time PCR. RESULTS Orthodontic tooth movement led to significant recruitment of OX6(+) cells and ED1(+) cells in the rat PDL. Double-immunofluorescence staining showed that some ED1(+) cells expressed pro-inflammatory factors of IL-1β and TNF-α in the PDL during orthodontic tooth movement. Real-time PCR analysis revealed that the expression levels of IL-1β (Il1b) and TNF-α (Tnf) mRNA gradually increased following its decrease after 1h of orthodontic tooth movement. These findings suggest that ED1(+) cells are involved in the expression of TNF-α and IL-1β and the subsequent regulation of bone resorption on pressure side. HSP27 (Hspb1) mRNA levels were significantly increased as compared with the control at 1h of the initial stage of treatment. CONCLUSION ED1(+) cells involved in the expression of TNF-α and IL-1β may play an important role in the initial reaction of the PDL and in the induction of the osteoclastic bone resorption during orthodontic tooth movement.

Fiber system degradation, and periostin and connective tissue growth factor level reduction, in the periodontal ligament of teeth in the absence of masticatory load

BACKGROUND AND OBJECTIVE The periodontal ligament (PDL), which is interposed between the alveolar bone and roots, supports teeth against mechanical stress. Periostin and connective tissue growth factor (CTGF) might play essential roles in maintaining PDL fiber integrity under mechanical stress. However, this relationship has not been studied at the protein and gene levels. Therefore, the aim of this study was to assess the PDL fiber system without masticatory load to determine the structural changes in the PDL in the absence of mechanical stress. MATERIAL AND METHODS The study included 45 Wistar male rats (12 wk of age) whose upper-right first molars were relieved from occlusion for 24 h, 72 h, 7 d or 21 d. The PDL was examined histologically, and changes in the gene and protein levels of periostin and CTGF were investigated. RESULTS The PDL space width was reduced significantly. Histologically, an initial reduction in the fiber number and thinning of PDL fibers were observed, followed by disarrangement of the PDL fibers and their attachments to the alveolar bone; finally, the PDL fibers lost their meshwork structure. Real-time RT-PCR results revealed sharp down-regulation of the periostin and CTGF mRNA levels at 24 and 72 h, respectively, which continued throughout the experiment. Immunohistochemical analysis revealed that periostin localized to both the cellular elements and the extracellular matrix, whereas CTGF localized only to the cellular elements. Periostin and CTGF immunoreactivities became very weak without masticatory load. CONCLUSION In the absence of mechanical stress, the PDL fiber system undergoes degradation concomitantly with a reduction in the periostin and CTGF levels in the PDL.

Laser capture microdissection of rat periodontal ligament for gene analysis

The periodontal ligament (PDL) is a connective tissue interposed between two hard tissues, viz., the root of a tooth and the alveolar bone, which makes it difficult to obtain directly. In the study reported here, PDL, subgingival connective tissue and pulp of rat molars were extracted directly by laser capture microdissection and the gene expression of TGF-β1 on the microdissected PDL was examined. The maxillae of rats were dissected and rapidly immersed in isopentane cooled with liquid nitrogen. Serial frontal sections of the rat first molar area were used for immunohistochemistry and for laser capture microdissection to localize the TGF-β1 gene. Gene expression and immunohistochemical localization of TGF-β1 also were examined in the pulp and subgingival connective tissues. TGF-β1 was located immunohistochemically in the fibroblasts in the PDL. A considerable amount of RNA was obtained by laser capture microdissection of these three tissues for analysis of gene expression. The reverse transcription- polymerase chain reaction demonstrated that glyceraldehyde 3-phosphate dehydrogenase was amplified in all three tissues, and that TGF-β1 was detected in the PDL. Laser capture microdissection makes it possible to analyze the gene expression of PDL and expression of TGF-β1in the PDL suggests that this gene could function in maintaining PDL.

Management of open bite that developed during treatment for internal derangement and osteoarthritis of the temporomandibular joint

This case report describes the orthodontic treatment performed for open bite caused by internal derangement (ID) and osteoarthritis (OA) of the temporomandibular joint (TMJ). A Japanese woman, aged 31 years and 11 months, referred to our department by an oral surgeon had an open bite with clockwise rotation of the mandible and degeneration of the condyle. The overbite was corrected through intrusion of the maxillary and mandibular molars using mini-screw implants to induce counterclockwise rotation of the mandible. Then, the mandibular second premolars were extracted and comprehensive orthodontic treatment was performed to establish a Class I molar relationship with distalization of the maxillary arch and to eliminate anterior crowding. Following treatment, her facial profile improved and a functional and stable occlusion was achieved without recurrence of the TMJ symptoms. These results suggest that orthodontic intrusion of the molars is one of the safer and less stressful alternatives for the management of open bite due to degeneration of the condyles caused by ID and OA of TMJ.

Mechanism of active eruption of molars in adolescent rats

The mechanism of active eruption of molars was examined in 36 male adolescent Wistar rats. Histological, histochemical [tetracycline (TC) labelling and alkaline phosphatase (ALP) activity], and immunohistochemical [transforming growth factor (TGF)-β1, -β2, and -β3] investigations were conducted of the rat molar areas. Real-time reverse transcription-polymerase chain reaction (RT-PCR) for mRNA of TGF-β was performed on the periodontal ligament (PDL) dissected out by laser capture microdissection. TC labelling lines showed that a considerable amount of bone formation occurred in the alveolar crest region, apical region, and intraradicular septum, indicating that the maxillary molars had moved downward. However, the periodontal fibres revealed a regular arrangement (alveolar crest, horizontal and oblique fibres) during the experimental period. This suggests that new formation of alveolar crest fibres and rearrangement of the periodontal fibres occurred in the PDL. ALP activity was intense on the bone surface and in the PDL. TGF-β1 was also detected in osteoblasts and fibroblasts but less so in cementoblasts. Real-time RT-PCR also demonstrated significant expression of mRNA of TGF-β1 in the PDL, indicating that TGF-β1 was involved in active eruption. These results suggest that active eruption occurs in adolescent rats and can be managed by TGF-β1.

Root resorption after experimental tooth movement using superelastic forces in the rat

The purpose of this study was to assess the rate of root resorption in relation to different magnitudes of continuous force during experimental tooth movement using nickel-titanium (NiTi) alloy wire. Four force magnitudes of 0.8, 1.6, 4, and 8 g were applied to the upper first molars of 75 male Wistar rats (300-320 g, 10-week-old) for 1, 7, 14, 21, and 28 days and compared with a control group without an orthodontic appliance. Light microscopic images of the compressed periodontal ligament (PDL) were processed by computer, and the ratio of the root resorption lacuna length to root surface length without the lacuna was analysed and statistically compared using Tukey-Kramer multiple comparison honestly significant difference test. The experimental groups with 4 and 8 g force showed undermining bone resorption with degenerating tissue and marked root resorption, the 1.6 g group showed only root resorption, while the 0.8 g group was similar to the control. Comparison of the ratios showed that the 0.8 g group was similar to the control with no significant difference. The ratio on day 28 in the 1.6 g group was larger than that in the 0.8 g and control groups, while on days 14, 21, and 28, the ratios in the 4 and 8 g groups were larger than those in the control (P < 0.01); these two experimental groups showed the same significant differences. It is suggested that significant root resorption occurs when the force magnitude exceeds 1.6 g in the rat upper first molar during tipping tooth movement by continuous force, and the amount of root resorption increases with serial force magnitudes from 0.8 to 4 g.

Extracellular HSP72 induces proinflammatory cytokines in human periodontal ligament fibroblast cells through the TLR4/NFκB pathway in vitro

OBJECTIVE The aim of the present study was to examine the effect of extracellular heat shock protein (HSP) 72 on human periodontal ligament fibroblast cells (hPDLFs) in vitro. DESIGN hPDLFs were stimulated by recombinant human HSP72 (rhHSP72). TAK-242 was used to inhibit toll-like receptor 4 (TLR4) activity. Interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-α mRNA levels were analyzed by real-time PCR and protein levels were analyzed by enzyme-linked immunosorbent assay. p65/RelA phosphorylation was analyzed by western blot. RESULTS IL-6, IL-8 and TNF-α mRNA and protein levels were significantly increased by rhHSP72 stimulation. These effects were inhibited by TAK-242 treatment. Additionally, p65/RelA phosphorylation was increased after 5-min rhHSP72 stimulation, which was inhibited by TAK-242 treatment. CONCLUSION Extracellular HSP72 induces proinflammatory cytokines through TLR4/NF-κB in hPDLFs.