Laser Photobiomodulation Over Teeth Subjected to Orthodontic Movement.

Background: Orthodontics of the 21st century requires aesthetic, painless, predictable, and quick treatments. This demand for faster results generated orthodontic movement acceleration protocols (OMAPs); among other OMAPs we present low-level laser (LLL) as a candidate. Objective: To evaluate levels of interleukin (IL)-1, IL-10, and type 1 collagen in the periodontal ligament of first molars of rats subjected to orthodontic traction with and without LLL irradiation, compared with untreated controls (CO), and to evaluate whether the dose of LLL used in this work is eligible as an OMAP. Materials and methods: A total of 35 male Wistar rats were distributed into three groups: group 1 NI (nonirradiated) n = 15, group 2 IR (laser irradiated using 5 J, 177 J/cm2, and 100 mW applied in contact to the vestibular mesial, vestibular distal, and palatal faces of gum tissue around molar region for 50 sec each point, for 3 consecutive days, immediately 24 and 48 h after orthodontic device placement.) n = 15, and group 3 CO n = 5; groups 1 and 2 were subjected to orthodontic force and each group was divided into three subgroups that were sacrificed after 3, 5, and 7 days, IL-1/10 and COL-1 levels were analyzed. Results: In the IR group, levels of IL-1/10 and COL-1 showed peak anticipation after LLL irradiation compared with those in the NI and CO groups. Conclusions: These results can also infer that this dose of LLL can be used as an OMAP.

Effects of electroacupuncture on experimental periodontitis in rats.

BACKGROUND: Acupuncture has shown the capability of modulating the immuno-inflammatory response of the host. This study aims to evaluate the effects of electroacupuncture (EA) on ligature-induced periodontitis in rats. METHODS: Thirty-two animals were divided into four groups: 1) control; 2) experimental periodontitis (EP); 3) sham-treated (EP/EA-sham); and 4) treated with EA (EP/EA). For the EP groups, a ligature was placed around the right mandibular first molars at day 1. Sessions of EA or EA-sham were assigned every other day. For EA treatment, large intestine meridian points LI4 and LI11 and stomach meridian points ST36 and ST44 were used. EA-sham was performed in off-meridian points. Animals were euthanized at day 11. Histomorphometric and microtomographic analyses were performed. Immunolabeling patterns for the receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) were assessed. Expressions of interleukin (IL)-1ß, matrix metalloproteinase (MMP)-8, IL-6, and cyclooxygenase (COX)-2 messenger RNAs (mRNAs) were evaluated by quantitative reverse transcription-polymerase chain reaction. Data were analyzed statistically (P <0.05, analysis of variance). RESULTS: Histomorphometric and microtomographic analyses demonstrated that group EP/EA presented reduced alveolar bone loss when compared to group EP (P <0.05). Reduced RANKL immunolabeling and fewer TRAP-positive multinucleated cells were observed in the EA-treated group in relation to group EP. No differences were observed in OPG expression among groups. EA treatment decreased the genic expression of IL-1ß and MMP-8 (P <0.05), increased the mRNA expression of IL-6 (P <0.05), and did not modify the genic expression of COX-2 in animals with EP (P >0.05). CONCLUSION: It can be concluded that EA reduced periodontal tissue breakdown and the expression of some proinflammatory mediators and a proresorptive factor in EP in rats.

Combining human periodontal ligament cell membrane chromatography with online HPLC/MS for screening osteoplastic active compounds from Coptidis Rhizoma.

We have developed an online analytical method that combines human periodontal ligament cell membrane chromatography (hPDLC/CMC) with high-performance liquid chromatography and mass spectrometry (LC/MS) for recognizing and identifying osteoplastic active components from Coptidis Rhizoma. Retention fractions on hPDLC/CMC were enriched onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using simvastatin (SIM) as a positive control, berberine from Coptidis Rhizoma was identified as the active component which could act on the hPDLC. The MTT colorimetric assay, alkaline phosphatase (ALP) activity, and staining tests revealed that berberine could promote hPDLC growth, increase the secretion of ALP in the culture medium, and enhance the formation of mineralized nodule, thus it is a potential osteoplastic ingredient. This hPDLC/CMC-online-LC/MS method can be applied for screening active components acting on hPDLC from traditional Chinese medicines exemplified by Coptidis Rhizoma and will be of great utility in drug discovery using natural medicinal herbs as a source of leading compounds.

Identification of N-methyl-D-aspartate receptor subunit in human periodontal ligament fibroblasts: potential role in regulating differentiation.

BACKGROUND: Periodontal ligament fibroblasts (PDLFs), which can be differentiated into osteoblasts, are crucial cells for the regeneration of the periodontal tissue. Although N-methyl-D-aspartate (NMDA) receptors were reported to be involved in bone formation by affecting osteoblasts, the existence and function of NMDA receptors in PDLFs have not been confirmed. The purpose of this study was to examine the expression of NMDA receptors and their role in human PDLFs. METHODS: Human PDLFs were cultured and evaluated to identify the subunits of NMDA receptors (NR) by reverse transcription-polymerase chain reaction, Western blot analysis, and immunocytochemistry. Then, the cells were assigned to four different groups: a control media group, a control media with NMDA receptor antagonist group, a differentiation media group, and a differentiation media with NMDA receptor antagonist group. Cell proliferation assay, alkaline phosphatase (ALP) activity analysis, and mineralization assay were performed to determine whether NMDA receptors affected the function of PDLFs. RESULTS: NR1, NR2B, and NR2D were detected in human PDLFs. There was no statistically significant difference in proliferation among the groups. However, the NMDA receptor antagonist-treated group showed a significant reduction in ALP activity (P <0.05). Moreover, the NMDA receptor antagonist-supplemented group presented no mineralization. CONCLUSIONS: This study revealed the existence of NMDA receptors in human PDLFs and specified their subunits. Moreover, NMDA receptors had a significant influence on the differentiation and mineralization of human PDLFs but did not affect their proliferation. These results suggest that NMDA receptors may play an important role in the differentiation and mineral tissue formation of human PDLFs.

Effects of Shuanghuangbu on the total protein content and ultrastructure in cultured human periodontal ligament cells.

BACKGROUND: Successful periodontal regeneration depends on the migration, proliferation and differentiation of periodontal ligament cells in periodontal defects. The total protein content and the ultrastructure demonstrate the metabolizability and activity of periodontal ligament cells. This study was conducted to observe the effects of Shuanghuangbu, a mixture of medicinal herbs, on the total protein content and the ultrastructure of human periodontal ligament cells. METHODS: Periodontal ligament cells were grown to confluence and then cultured in Dulbecco's modified eagle medium (DMEM) supplemented with Shuanghuangbu over the concentration range of 0 to 1000 microg/ml. The total protein content in cultured cells was determined by using Coommasie brilliant blue technique. Periodontal ligament cells were incubated in 0 and 100 microg/ml Shuanghuangbu decoction for 5 days, then observed through transmission electron microscope. RESULTS: The total protein content of human periodontal ligament cells increased in each experiment group added 10 - 1000 microg/ml Shuanghuangbu respectively, and the effect of 100 microg/ml was excellent. Under the transmission electron microscope, there were more rough endoplasmic reticulums and mitochodrias in the experiment group than those in the control group. CONCLUSION: Shuanghuangbu stimulates the protein synthesis of human periodontal ligament cells and improves human periodontal ligament cells' metabolizability and activity.

cDNA cloning of S100 calcium-binding proteins from bovine periodontal ligament and their expression in oral tissues.

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of beta-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

New findings in the degenerating tissues of the periodontal ligament during experimental tooth movement.

The degenerating tissues found in rat periodontal ligaments during tooth movement were examined morphologically, histochemically, and elementally, with decalcified and unfixed, undecalcified frozen sections. There were two types of degenerating tissues found in the compressed periodontal ligaments: One (type A tissue) was stained differently from collagen and the other (type B tissue) showed the same color as collagen. Type A tissue also showed the deposition of fibrin in Martius scalet blue and Weigert stain. The electron micrograph also showed the deposition of fibrin in type A tissue. No collagen fibers with typical bandings were seen in either tissue. The digestion experiment showed that type A tissue was digested by trypsin but not type B, whereas most of type B tissue was digested by collagenase but not type A. The backscattered electron image by scanning electron microscopy of type A tissue of the unfixed undecalcified frozen sections showed the presence of many small pieces. The elemental analysis of the pieces showed high peaks of phosphorous and calcium. These results indicate that collagen degradation, fibrin deposition, and calcification occurred in the degenerating tissues, especially in type A tissue during the experimental tooth movement.

The effect of glycosylaminoglycans on the mineralization of sheep periodontal ligament in vitro.

The effect of removal of glycosylaminoglycans on the mineralization of sheep periodontal ligament was determined using enzyme digests followed by incubation in solutions supersaturated with respect to hydroxyapatite at pH 7.4. TEM revealed that control periodontal ligament remained unmineralized. However, tissue from which glycosylaminoglycans had been removed contained plate-like crystals arranged parallel to and within the collagen fibrils. Electron probe and electron diffraction studies suggested that the crystals were apatitic with a similar order of crystallinity to dentine, and a Ca:P ratio of 1.61. In addition, the glycosylaminoglycan content of periodontal ligament, cementum and alveolar bone was compared using cellulose acetate electrophoresis. Periodontal ligament contained predominantly dermatan sulfate while cementum and alveolar bone contained mostly chondroitin sulfate. A role for glycosylaminoglycans in maintaining the unmineralized state of the periodontal ligament is suggested. Control of expression of specific proteoglycan species on a spatially restricted basis is presumably central to this role.

In vitro formation of mineralized nodules by periodontal ligament cells from the rat.

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical study of human periodontal ligament: preparation of cell attachment materials induced by pulsed electromagnetic fields.

The periodontium, especially the periodontal ligament and alveolar bone, are tissues constantly subjected to physical stress such as occlusion and mastication. This study was designed to explore the effect of the pulsed electromagnetic fields (PEMF) on the cell attachment and the spread of human periodontal ligament fibroblasts (HPLF) and rat osteoblasts (ROB). PEMF are categorized as one type of mechanical stress. HPLF were obtained by the explantation method described by Saito et al. They were then subcultured in Dulbecco's modified Eagle's medium (D-MEM) and supplemented with 2 mg/ml dialyzed fetal calf serum protein (FCSP), 50 micrograms/ml ascorbic acid and penicillin/streptomycin after trypsinization. ROB were isolated from a two-day-old rat calvaria by the sequential bacterial collagenase digestion method described by Dziak and Brand and were subcultured in D-MEM supplemented with FCSP, ascorbic acid and penicillin/streptomycin. After the confluent HPLF were cultured with serum-free MCDB 107 medium, the quiescent HPLF were exposed with or without PEMF for 24 hr. This was followed by the collection of the control conditioned medium (C-CM) and PEMF exposed conditioned medium (PEMF-CM). The cell attachment assay was performed so that the hydrophobic 24 multiwells were coated with the whole conditioned medium or fractionated conditioned medium by a PO-60K column. After coating, heat inactivated BSA blocked nonspecific sites for cell adhesion, and 3H-TdR labeled HPLF or ROB were cultured on the precoated wells. The activity of cell attachment and spreading was determined by the radioactivity of 3H-TdR using a scintillation counter. The characters of cell attachment factors derived from HPLF were hydrophobic, heat labile and proteolytic enzyme digestible. In addition, the fractionated PEMF-CM enhanced the spreading activity of ROB. PEMF induced the 10 KDa which can enhance the HPLF and ROB spreading. Therefore, the cell attachment and spreading factors secreted by HPLF exposed with PEMF may regulate HPLF and also ROB.