Effect of Low Intensity Laser Therapy (LILT) on MMP-9 expression in gingival crevicular fluid and rate of orthodontic tooth movement in patients undergoing canine retraction: A randomized controlled trial.

INTRODUCTION: Low Intensity Laser Therapy (LILT) has been shown to increase the rate of tooth movement. Since its use in orthodontics as a method of acceleration there has been a variety of views regarding its mode of action. MMP-9 is a known bone resorption factor studied in Bone remodelling. The aim of this study was to know the effect of LILT on rate of tooth movement and expression of MMP-9 in GCF. MATERIALS AND METHODS: Ten patients (3 males and 7 females) who required maxillary first premolar extraction for routine orthodontic treatment were recruited. The individual canine retraction was studied, and the side of the experimental canine was randomly selected. The laser regimen was followed on the 1st, 3rd, 5th, 7th, 14th and then 15th days consecutively. GCF was collected at baseline, 14th day, 3 months and at the end of canine retraction on experimental side and MMP-9 was estimated quantitatively using a standard ELISA kit. RESULTS: The average increase in rate of tooth movement on experimental side at 3 months was 44% and MMP-9 concentration was also high. At the end of canine retraction (4.5 months) in the experimental group the average rate increase was 38% with MMP-9 concentrations similar in both the experimental and control group. CONCLUSIONS: LILT increases the rate of tooth movement. LILT also has an effect of bio-stimulation as depicted by rise in MMP-9 concentrations in GCF. However, this bio-stimulatory effect is restricted to the initial part of the tooth movement.

Establishment of an orthodontic retention mouse model and the effect of anti-c-Fms antibody on orthodontic relapse.

Orthodontic relapse after orthodontic treatment is a major clinical issue in the dental field. However, the biological mechanism of orthodontic relapse is still unclear. This study aimed to establish a mouse model of orthodontic retention to examine how retention affects the rate and the amount of orthodontic relapse. We also sought to examine the role of osteoclastogenesis in relapse using an antibody to block the activity of M-CSF, an essential factor of osteoclast formation. Mice were treated with a nickel-titanium closed-coil spring that was fixed between the upper incisors and the upper-left first molar to move the first molar in a mesial direction over 12 days. Mice were randomly divided into three groups: group 1, no retention (G1); group 2, retention for 2 weeks (G2); and group 3, retention for 4 weeks (G3). In G2 and G3, a light-cured resin was placed in the space between the first and second molars as a model of retention. Orthodontic relapse was assessed by measuring changes in the dimensions of the gap created between the first and second molars. To assess the activity and role of osteoclasts, mice in G3 were injected with anti-c-Fms antibody or PBS, and assessed for changes in relapse distance and rate. Overall, we found that a longer retention period was associated with a slower rate of relapse and a shorter overall amount of relapse. In addition, inhibiting osteoclast formation using the anti-c-Fms antibody also reduced orthodontic relapse. These results suggest that M-CSF and/or its receptor could be potential therapeutic targets in the prevention and treatment of orthodontic relapse.

Microanatomical changes and biomolecular expression at the PDL-entheses during experimental tooth movement.

The novel aspect of this study was to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL)-space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week-old male C57BL/6 mice (N = 5) were experimentally translated for 2 weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship's resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurred as an attempt to restore PDL-space of the load-bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs provided insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally induced resorption, and naturally occurring mineral resorption to experimentally induced formation at the enthesial sites to permit tooth translation.

Osteocyte regulation of orthodontic force-mediated tooth movement via RANKL expression.

Orthodontic tooth movement is achieved by the remodeling of the alveolar bone surrounding roots of teeth. Upon the application of orthodontic force, osteoclastic bone resorption occurs on the compression side of alveolar bone, towards which the teeth are driven. However, the molecular basis for the regulatory mechanisms underlying alveolar bone remodeling has not been sufficiently elucidated. Osteoclastogenesis is regulated by receptor activator of nuclear factor-κB ligand (RANKL), which is postulated to be expressed by the cells surrounding the tooth roots. Here, we show that osteocytes are the critical source of RANKL in alveolar bone remodeling during orthodontic tooth movement. Using a newly established method for the isolation of periodontal tissue component cells from alveolar bone, we found that osteocytes expressed a much higher amount of RANKL than other cells did in periodontal tissue. The critical role of osteocyte-derived RANKL was confirmed by the reduction of orthodontic tooth movement in mice specifically lacking RANKL in osteocytes. Thus, we provide in vivo evidence for the key role of osteocyte-derived RANKL in alveolar bone remodeling, establishing a molecular basis for orthodontic force-mediated bone resorption.

Sclerostin Promotes Bone Remodeling in the Process of Tooth Movement.

Tooth movement is a biological process of bone remodeling induced by mechanical force. Sclerostin secreted by osteocytes is mechanosensory and important in bone remodeling. However, little is known regarding the role of sclerostin in tooth movement. In this study, models of experimental tooth movement were established in rats and mice. Sclerostin expression was investigated with immunohistochemistry staining, and osteoclastic activity was analyzed with tartrate-resistant acid phosphatase (TRAP) staining. MLO-Y4 osteocyte-like cells underwent uniaxial compression and tension stress or were cultured in hypoxia conditions. Expression of sclerostin was assessed by RT-qPCR and ELISA. MLO-Y4 cells were cultured with recombinant human sclerostin (rhSCL) interference and then co-cultured with RAW264.7 osteoclast precursor cells. Expressions of RANKL and OPG were analyzed by RT-qPCR, and osteoclastic activity was assessed by TRAP staining. During tooth movement, sclerostin was expressed differently in compression and tension sites. In SOST knock-out mice, there were significantly fewer TRAP-positive cells than in WT mice during tooth movement in compression sites. In-vitro studies showed that the expression of sclerostin in MLO-Y4 osteocyte-like cells was not different under a uniaxial compression and tension force, whereas hypoxia conditions significantly increased sclerostin expression in MLO-Y4 cells. rhSCL interference increased the expression of RANKL and the RANKL/OPG ratio in MLO-Y4 cells and the osteoclastic induction ability of MLO-Y4 cells in experimental osteocyte-osteoclast co-culture. These data suggest that sclerostin plays an important role in the bone remodeling of tooth movement.

Combined effects of proinflammatory cytokines and intermittent cyclic mechanical strain in inhibiting osteogenicity in human periodontal ligament cells.

Mechanical strain plays an important role in bone formation and resorption during orthodontic tooth movement. The mechanism has not been fully studied, and the process becomes complex with increased amounts of periodontal patients seeking orthodontic care. Our aims were to elucidate the combined effects of proinflammatory cytokines and intermittent cyclic strain (ICS) on the osteogenic capacity of human periodontal ligament cells. Cultured human periodontal ligament cells were exposed to proinflammatory cytokines (interleukin-1ß 5 ng/mL and tumor necrosis factor-α 10 ng/mL) for 1 and 5 days, and ICS (0.5 Hz, 12% elongation) was applied for 4 h per day. The autocrine of inflammatory cytokines was measured by enzyme-linked immunosorbent assay. The expression of osteoblast markers runt-related transcription factor 2 and rabbit collagen type I was determined using real-time polymerase chain reaction and Western blot. The osteogenic capacity was also detected by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. We demonstrated that ICS impaired the osteogenic capacity of human periodontal ligament cells when incubated with proinflammatory cytokines, as evidenced by the low expression of ALP staining, low ALP activity, reduced alizarin red staining, and reduced osteoblast markers. These data, for the first time, suggest that ICS has a negative effect on the inductive inhibition of osteogenicity in human PDL cells mediated by proinflammatory cytokines.

Nanoemulgel (NEG) of Ketoprofen with eugenol as oil phase for the treatment of ligature-induced experimental periodontitis in Wistar rats.

AIM: The aim of the novel study was to check the efficacy of a locally applied 2%w/w nanoemulgel (NEG) of Ketoprofen (KP) in preventing the periodontitis, and was also checked NEG without KP to ensure the effect of eugenol in NEG as an oil phase. DESIGN: For experimentally induced periodontitis, sterile silk ligatures (3/0) were placed around the crevices of the first left lower molar teeth of the male Wistar rats. During 8 weeks, all rats were fed with 10%w/v sucrose solution. The experimental assessment was carried out at 11 d after treatment of experimental periodontal disease (EPD) rats by various clinical parameters like gingival index (GI), tooth mobility (TM), alveolar bone loss (ABL), histological analysis, detection of TNF-α, and IL-1ß in gingival tissue by ELISA and the roughness were measured by atomic force microscopy (AFM) in tapping modes. RESULTS: After treatment, comparison studies with EPD were performed. NEG loaded with KP prevents significantly (p < 0.05) various parameters (GI, TM, and ABL), which were responsible for periodontitis. The histopathology of the periodontium showed that Group 3 (NEG loaded with KP) had a more significant reduction in inflammatory cell infiltration, alveolar bones resorption, and cementum (p < 0.05). In the topographical images, significant reduction in roughness of NEG loaded with KP was observed in comparison with EPD without treatment. CONCLUSION: The study revealed the great synergistic potential of the combined NEG of an anti-inflammatory drug KP along with eugenol as the oil phase, which have potential antibacterial, analgesic, and anesthetic properties to combat periodontal disease.

Periodontal CGRP contributes to orofacial pain following experimental tooth movement in rats.

Calcitonin-related gene peptide (CGRP) plays an important role in orofacial inflammatory pain. The aim of this study was to determine whether periodontal CGRP contributes to orofacial pain induced by experimental tooth movement in rats. Male Sprague-Dawley rats were used in this study. Closed coil springs were used to deliver forces. Rats were euthanized on 0d, 1d, 3d, 5d, 7d, and 14d following experimental tooth movement. Then, alveolar bones were obtained for immunostaining of periodontal tissues against CGRP. Two hours prior to euthanasia on each day, orofacial pain levels were assessed through rat grimace scale. CGRP and olcegepant (CGRP receptor antagonist) were injected into periodontal tissues to verify the roles of periodontal CGRP in orofacial pain induced by experimental tooth movement. Periodontal CGRP expression levels and orofacial pain levels were elevated on 1d, 3d, 5d, and 7d following experimental tooth movement. The two indices were significantly correlated with each other and fitted into a dose-response model. Periodontal administration of CGRP could elevate periodontal CGRP expressions and exacerbate orofacial pain. Moreover, olcegepant administration could decrease periodontal CGRP expressions and alleviate orofacial pain. Therefore, periodontal CGRP plays an important role in pain transmission and modulation following experimental tooth movement. We suggest that it may participate in a positive feedback aiming to amplify orofacial pain signals.

Expression of HMGB1 in the periodontal tissue subjected to orthodontic force application by Waldo's method in mice.

Recent studies indicate that high mobility group box protein 1 (HMGB1) originating from periodontal ligament (PDL) cells can be a potential regulator in the process of orthodontic tooth movement and periodontal tissue remodeling. The aim of this study is to investigate HMGB1 expression in periodontal tissue during orthodontic tooth movement in mice according to Waldo's method. Six 7-week-old C57BL6 mice were used in these experiments. The elastic band was inserted into the teeth space between the right first and second maxillary molars. After 3 days of mechanical loading, mice were fixed with transcardial perfusion of 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and the maxillary was extracted for histochemical analyses. The histological examination revealed local PDL tear at the tension side and the formation of extensive cell-free hyaline zones at the compression side. The immunolocalization of HMGB1 was significantly presented at tension side of PDL, apical area and dental pulp, whereas at the compression side of PDL, the labeling of HMGB1 was almost undetectable as the presence of hyaline zone. Taken together, we concluded that the orthodontic tooth movement by Waldo's method leads to histological changes and HMGB1 expression pattern that differ from those of coil spring method, including PDL tear and extensive hyaline zone which may severely destroy periodontal tissue and in turn impede tooth movement.

Low-intensity pulsed ultrasound accelerates tooth movement via activation of the BMP-2 signaling pathway.

The present study was designed to determine the underlying mechanism of low-intensity pulsed ultrasound (LIPUS) induced alveolar bone remodeling and the role of BMP-2 expression in a rat orthodontic tooth movement model. Orthodontic appliances were placed between the homonymy upper first molars and the upper central incisors in rats under general anesthesia, followed by daily 20-min LIPUS or sham LIPUS treatment beginning at day 0. Tooth movement distances and molecular changes were evaluated at each observation point. In vitro and in vivo studies were conducted to detect HGF (Hepatocyte growth factor)/Runx2/BMP-2 signaling pathways and receptor activator of NFκB ligand (RANKL) expression by quantitative real time PCR (qRT-PCR), Western blot and immunohistochemistry. At day 3, LIPUS had no effect on the rat orthodontic tooth movement distance and BMP-2-induced alveolar bone remodeling. However, beginning at day 5 and for the following time points, LIPUS significantly increased orthodontic tooth movement distance and BMP-2 signaling pathway and RANKL expression compared with the control group. The qRT-PCR and Western blot data in vitro and in vivo to study BMP-2 expression were consistent with the immunohistochemistry observations. The present study demonstrates that LIPUS promotes alveolar bone remodeling by stimulating the HGF/Runx2/BMP-2 signaling pathway and RANKL expression in a rat orthodontic tooth movement model, and LIPUS increased BMP-2 expression via Runx2 regulation.

Biomarkers of periodontal tissue remodeling during orthodontic tooth movement in mice and men: overview and clinical relevance.

Biologically active substances are expressed by cells within the periodontium in response to mechanical stimuli from orthodontic appliances. Several possible biomarkers representing biological modifications during specific phenomena as simile-inflammatory process, bone resorption and formation, periodontal ligament changes, and vascular and neural responses are proposed. Citations to potentially published trials were conducted by searching PubMed, Cochrane databases, and scientific textbooks. Additionally, hand searching and contact with experts in the area were undertaken to identify potentially relevant published and unpublished studies. Selection criteria were as follows: animal models involving only mice and rats undergoing orthodontic treatment; collection of gingival crevicular fluid (GCF) as a noninvasively procedure for humans; no other simultaneous treatment that could affect experimental orthodontic movement. The data suggest that knowledge of the remodeling process occurring in periodontal tissues during orthodontic and orthopedic therapies may be a clinical usefulness procedure leading to proper choice of mechanical stress to improve and to shorten the period of treatment, avoiding adverse consequences. The relevance for clinicians of evaluating the rate of some substances as valid biomarkers of periodontal effects during orthodontic movement, by means of two models of study, mice and men, is underlined.

The effect of antioxidants on the production of pro-inflammatory cytokines and orthodontic tooth movement.

Orthodontic force causes gradual compression of the periodontal ligament tissues, which leads to local hypoxia in the compression side of the tissues. In this study, we investigated whether antioxidants exert a regulatory effect on two factors: the expression of pro-inflammatory cytokines in human periodontal ligament fibroblasts (PDLFs) that were exposed to mechanical compression and hypoxia and the rate of orthodontic tooth movement in rats. Exposure of PDLFs to mechanical compression (0.5-3.0 g/cm(2)) or hypoxic conditions increased the production of intracellular reactive oxygen species. Hypoxic treatment for 24 h increased the mRNA levels of IL-1ß, IL-6 and IL-8 as well as vascular endothelial growth factor (VEGF) in PDLFs. Resveratrol (10 nM) or N-acetylcysteine (NAC, 20 mM) diminished the transcriptional activity of hypoxiainducible factor-1 and hypoxia-induced expression of VEGF. Combined treatment with mechanical compression and hypoxia significantly increased the expression levels of IL-1ß, IL-6, IL-8, TNF-α and VEGF in PDLFs. These levels were suppressed by NAC and resveratrol. The maxillary first molars of rats were moved mesially for seven days using an orthodontic appliance. NAC decreased the amount of orthodontic tooth movement compared to the vehicle-treated group. The results from immunohistochemical staining demonstrated that NAC suppressed the expression of IL-1ß and TNF-α in the periodontal ligament tissues compared to the vehicle-treated group. These results suggest that antioxidants have the potential to negatively regulate the rate of orthodontic tooth movement through the down-regulation of pro-inflammatory cytokines in the compression sides of periodontal ligament tissues.

Early responses of periodontal ligament cells to mechanical stimulus in vivo.

Previous studies have indicated that human periodontal ligament cells undergo osteoblastic differentiation via the ERK pathway under mechanical stress in vitro. This study aimed to verify this principle in vivo. The right upper first molars of 25 anesthetized rats were loaded with constant forces of 0.1 N for up to 8 hrs. The untreated contralateral side served as a control. Paraffin-embedded sections were analyzed by immunohistochemistry for proliferating cell nuclear antigen (PCNA), runt-related transcription factor 2 (Runx2/Cbfa1), and phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2). In selected areas under tension, the proportions of Runx2-positive and pERK1/2-positive cells increased within 8 hrs of loading, whereas these proportions in selected areas under pressure were significantly lower than those in control teeth. Moreover, there were no significant changes in the number of PCNA-positive cells. Thus, mechanical stimulus up-regulates Runx2, and this regulation may be achieved via the ERK pathway.

Experimental arthritis in rats induces biomarkers of periodontitis which are ameliorated by gene therapy with tissue inhibitor of matrix metalloproteinases.

BACKGROUND: Periodontal disease (PD) and rheumatoid arthritis (RA) share many common pathophysiologic features, but a clinical relationship between the two conditions remains controversial, in part because of the confounding effects of anti-inflammatory drug therapy universally used in the latter disease. To further explore this issue, inflammatory arthritis was induced in rats to determine the effect on gingival biomarkers of inflammation and tissue destruction and to investigate the effect of a therapeutic intervention devoid of conventional anti-inflammatory properties. METHODS: Adjuvant arthritis (AA) was induced in Lewis male rats by injecting mycobacterium cell wall in complete Freund's adjuvant using standard techniques. One group of animals was treated by induction of systemic tissue inhibitor of matrix metalloproteinases (TIMP-4). At 3 weeks, arthritis severity was recorded and both paw and gingival tissues were collected for matrix metalloproteinase activity (MMP) and cytokine analysis. In addition, the maxillary jaws were removed for assessment of periodontal bone loss. RESULTS: The development of arthritis was associated with elevated joint tissue MMPs, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta levels compared to control rats. In the gingival tissue of the untreated arthritic rats, gelatinase, collagenase, TNF-alpha, and IL-1beta were also elevated compared to control rats. Periodontal bone loss and tooth mobility were also increased significantly (P <0.05) in untreated arthritic rats. All parameters improved after TIMP-4 gene therapy. CONCLUSIONS: To our knowledge, this is the first study to report an association between experimental systemic arthritis in rats and elevated gingival tissue MMPs, cytokine levels, and periodontal disease. Reversal of these changes with TIMP-4 gene therapy strengthens the pathophysiologic correlation between systemic and local disease.

Expression of mRNAs encoding for alpha and beta integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts.

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and alpha and beta integrin subunits. Both cell lines reacted by inducing the expression of the mRNAs encoding for MMP-1, MMP-2, TIMP-1, and TIMP-2, while other mRNAs did not vary (MT1-MMP, TIMP-3) or were not expressed (MMP-9). PDLs expressed selectively the mRNAs encoding for alpha4 and alphav, with no difference measurable under stretching, while the mRNAs encoding for alpha6 and beta1 were increased and the one encoding for alpha5 was decreased. HGFs increased the mRNAs encoding for alpha2, alpha6, beta1, and beta3 and decreased the one encoding for alpha3. Analysis of our data indicated that stretched HGFs and PDLs induced the same pattern of mRNAs encoding for MMPs and TIMPs but differed for those encoding various integrin subunits, known to act as protein receptors in mechanotransduction.

Effects of morphine on the distribution of Fos protein in the trigeminal subnucleus caudalis neurons during experimental tooth movement of the rat molar.

The present study was undertaken to disclose temporal changes in the distribution of Fos-like immunoreactive (-IR) neurons in the trigeminal subnucleus caudalis (SpVc), one of the important relay nuclei for processing the nociceptive information from the oro-facial regions, following induction of experimental tooth movement in rat upper molars. Furthermore, the effect of morphine and naloxone on the levels of Fos-IR neurons in the SpVc was examined. The experimental tooth movement was induced by insertion of an elastic rubber between the first and second upper molars. In normal animals, Fos-IR neurons were rarely observed in the SpVc. Immediately after insertion of the elastic band, the distribution of Fos-IR neurons was comparable to that observed in normal animals. The number of Fos-IR neurons increased significantly from 1 to 4 h following the induction of experimental tooth movement, reaching a maximum at 2 h, and then decreasing gradually. Most of the neurons were localized in the dorsomedial portion of the superficial layers of the ipsilateral SpVc near the obex, but a few were observed at the ventral portion of the SpVc. The neurons at the superficial layers and ventral portion of the contralateral SpVc also showed Fos-like immunoreactivity, but their numbers were significantly smaller than those on the ipsilateral side. Pretreatment with morphine (3 and 10 mg/kg, i.p.) significantly reduced the induction of Fos-IR neurons at the superficial layers of the ipsilateral SpVc in a dose-dependent manner, and its effect was antagonized by the subsequent treatment of naloxone (2 mg/kg, i.p.). Naloxone pretreatment enhanced the expression of Fos-IR neurons on the ipsilateral SpVc. The present results of a reduction of Fos-IR neurons by morphine pretreatment suggest that the induction of Fos-IR neurons may be due to the noxious stimulation caused by induction of experimental tooth movement.

Levels of interleukin-1 beta and interleukin-8 in gingival crevicular fluids in adult periodontitis.

Recent in vitro findings indicate that cytokines represent an important pathway of connective tissue destruction in human periodontitis. The biological effects of interleukin-1 beta (IL-1 beta) and interleukin-8 (IL-8) are relevant in this regard, and the objective of this study was to compare the levels of these molecules in gingival crevicular fluids (GCF) from patients with adult periodontitis (experimental group) and from individuals with clinically healthy gingiva (control group). GCF was collected for 30 seconds using a periopaper strip and the volume of the sample determined. Following elution of the fluid, assays for IL-1 beta and IL-8 were carried out by ELISA. The concentrations (ng/ml) of cytokines were calculated in the original volume of GCF on each strip. The total amounts (pg/site) of cytokines were expressed as the concentrations multiplied by volumes of GCF: The total amounts of IL-1 beta and IL-8 of the experimental group were significantly higher than the control group. The total amounts of both cytokines were markedly reduced following phase 1 periodontal treatment. The clinical parameters were positively related to the total amounts of IL-1 beta and IL-8. IL-1 beta concentrations and total amounts were also positively related to IL-8 suggesting that the GCF IL-8 levels are influenced by local IL-1 beta activities. These data indicate that the total amounts of IL-1 beta and IL-8 exhibited dynamic changes upon severity of periodontal disease. The levels of IL-1 beta and IL-8 in GCF are valuable in detecting the inflammation of periodontal tissue.

Preparation of unfixed and undecalcified frozen sections of adult rat periodontal ligament during experimental tooth movement.

The upper first molars of adult male rats were moved for 7 days and unfixed, undecalcified frozen sections of the molar periodontal ligament were prepared and observed. The upper jaws of the rats were immersed rapidly in liquid nitrogen and sectioned with a cryostat using a super hard knife. Five micrometer serial sections were cut, collected, freeze-dried and observed with both light and scanning electron microscopy. Electron probe microanalysis (EPMA) was also performed on the sections. On the tension side of the periodontal ligament, periodontal fibers were stretched and the osteoblasts were aligned on the osteoid, which showed metamasia with the toluidine blue stain. On the pressure side where the periodontal ligament was extremely compressed, tissue degeneration was caused by tooth movement and the osteoclasts were observed on the bone surface adjacent to the degenerating tissues. Scanning electron microscopy revealed a network arrangement of the collagen fiber bundles on the tension side, but not on the pressure side of the periodontal ligament. The spectrum obtained from EPMA of the osteoid demonstrated X-ray (Ka) peaks of Na, P, S, K and Ca.

Site-specific expression of mRNAs for osteonectin, osteocalcin, and osteopontin revealed by in situ hybridization in rat periodontal ligament during physiological tooth movement.

We investigated the gene expression for non-collagenous proteins in periodontal ligament (PDL) by in situ hybridization histochemistry with a non-radioisotopic probe with cRNAs for osteocalcin (Osc), osteonectin (Osn), and osteopontin (Opn) in rat maxillary dento-alveolar unit containing molars and intact PDL. A highly intense positive signal for Osn and Osc mRNAs was expressed at all distal surfaces of the interradicular septum of buccal roots of the upper second molar in 7-week-old Sprague-Dawley male rats. Cells showing positive signals for Osn and Osc mRNAs were osteoblasts and osteoprogenitor cells. The distribution of Opn mRNA-positive signal was demonstrable at the mesial surface of the interradicular septum of buccal roots, where physiological bone resorption was specifically restricted during physiological tooth movement. Opn mRNA was expressed in cells on the bone resorption surface, including osteoclasts, and osteocytes. A moderately intense positive signal for Osn mRNA was distributed in fibroblasts throughout the ligament. Odontoblasts and pre-mature odontoblasts exhibited a strong signal for Osn and Osc mRNA. Cementoblasts and cementocytes were positive for Osn, Osc, and Opn mRNAs. These findings suggest physiological roles of Osc, Osn, and Opn in bone remodeling, PDL remodeling, dentinogenesis, and cementogenesis.

A cross-sectional study on osteocalcin levels in gingival crevicular fluid from periodontal patients.

The purpose of the present study was to determine the levels of osteocalcin, a bone specific matrix protein, in gingival crevicular fluid (GCF) from periodontal disease patients and to investigate the relationship between GCF osteocalcin levels and clinical parameters. Nineteen initial visit patients, 5 patients with gingivitis and 14 patients with adult periodontitis, participated in this study. The clinical parameters including probing depth, attachment level, gingival index, and tooth mobility were recorded following careful sampling of GCF with a filter paper strip harvested for 3 minutes. Osteocalcin adsorbed on a strip was extracted in a plastic tube containing 150 microliters of 10 mM sodium phosphate buffer (pH 6.5). GCF osteocalcin was determined by a newly-developed, high sensitive enzyme immunoassay which could recognize the N-terminal 20 residue peptide. In gingivitis patients, no significant amounts of osteocalcin were detected. In periodontitis patients, on the other hand, osteocalcin levels were detected, ranging between 0 and 540 pg/tube and positively correlated with these clinical parameters (P < 0.01). Moreover, in several sites in GI = 3 group, extremely higher levels of GCF osteocalcin were detected. These results strongly suggest that in addition to the presence of GCF osteocalcin the levels of osteocalcin may reflect the degree of the periodontal inflammation at the sampled sites.