Prostaglandin E2 affects osteoblast biology in a dose-dependent manner: an in vitro study.

OBJECTIVE: This study aimed to determine in vitro how exogenous PGE(2) affects the expression of genes in cultured osteoblasts by relative quantitation PCR. DESIGN: Cultured osteoblasts were exposed to 10(-3)M, 10(-5)M or 10(-7)M PGE(2) over 5, 10, 15 and 20 days. RESULTS: RANKL expression was higher after 5 days of exposure (p<0.05), but thereafter reduced in those treated with the two lower doses of PGE(2) (p<0.01). RANKL/OPG ratio reported in favour of OPG gene expression and alkaline phosphatase gene expression increased in osteoblasts exposed to the two lower doses of the eicosanoid after 15 days. Conversely, prostaglandin E synthase, a cytokine produced during PGE(2) synthesis, gene expression was significantly reduced at 15 and 20 days (p<0.01 and 0.05 respectively). The results from this study add to the current knowledge of the mechanisms by which PGE(2) modulates the osteoblast biology in a dose-dependent manner. CONCLUSIONS: It is proposed that PGE(2)at a low dose switch osteoblast's biology in favour of bone apposition by: first, inducing a significantly higher OPG gene expression overwhelming RANKL gene expression; second, reducing PGEs synthesis; and third, increasing ALP gene expression. An opposite effect is expected when the concentration of the eicosanoid overpass certain levels.

Prostaglandin E2 enhances transforming growth factor-beta 1 and TGF-beta receptors synthesis: an in vivo and in vitro study.

The aims of this study were to determine how Prostaglandin E2 (PGE2) locally applied affected the immunodistribution of latent transforming growth factor-beta 1 (TGF-beta1), and how the eicosanoid modified TGF-beta1 release and TGF-beta receptors gene expression in cultured osteoblasts. PGE2 locally delivered on the rat mandible at doses of 0.1 and 0.05 mg/day, but not 0.025 mg/day, over 20 days significantly increased latent TGF-beta1 immunodistribution (P<0.001), comparing with a placebo-treated group. Cultured osteoblasts stimulated with 10(-5) or 10(-7)M PGE2 significantly varied the level of activated TGF-beta1 released into supernatants at different experimental periods compared with negative and positive controls. TGF-beta receptor type I gene expression was significantly increased in osteoblasts (P<0.01) after 10 days of treatment with 10(-5) and 10(-7)M PGE2, whereas 10(-3) M PGE2 produced the opposite effect. It is concluded that PGE2 may stimulate bone deposition by affecting TGF-beta pathway. This effect on the pathway appears to be dose-dependent.

Local application of prostaglandin E2 reduces trap, calcitonin receptor and metalloproteinase-2 immunoreactivity in the rat periodontium.

It has been shown that prostaglandin E2 (PGE2) locally released adjacent to the mandible over a 20-day period increases alveolar bone area, in part, due to a reduction in the percentage of eroded surface. To determine the effect of PGE2 on alveolar bone resorption, left mandibles from 24 Lewis rats were treated over a 20-day period with a local application of PGE2 (0.1, 0.05 or 0.025 mg/day) or placebo. The right side served as the non-treated matched control. Tissue sections were stained for tartrate resistant acid phosphatase (TRAP) calcitonin receptor (CTR) and metalloproteinase-2 (MMP-2). Matched samples were analysed by Wilcoxon matched pairs test and, a non-parametric one-way analysis of variance compared groups of treatment. Those tissues treated with PGE2 at doses of 0.1 and 0.05 mg/day showed significantly reduced numbers of TRAP and CTR-positive multinucleated cells compared with matched controls (p<0.005), as well as significantly reduced numbers of TRAP- and CTR-positive multinucleated cells when compared with the placebo-treated group (p<0.001). The number of periodontal ligament cells expressing MMP-2 was also significantly reduced in tissues treated with the two higher doses of PGE2 (p<0.001) comparing with both matched controls and the placebo-treated group. Following a 20-day period, locally released PGE2 at doses of 0.1 and 0.05 mg/day appears to affect alveolar bone resorption in the periodontium of rats, as the number of multinucleated cells expressing TRAP and CTR are significantly reduced. Furthermore, the same doses of PGE2 also significantly reduced the expression of MMP-2 by the periodontal cells.

Prostaglandin E2 enhances alveolar bone formation in the rat mandible.

Prostaglandin E2 (PGE2) induces bone formation in stress-bearing bones. The mandible, a stress-bearing bone, is loaded daily during mastication. The aim of this study was to determine if PGE2 delivered locally to the mandible over 20 days enhances alveolar bone deposition. In 18 Lewis rats, controlled-release pellets containing PGE2 were implanted on the buccal aspect on the left-hand side of the mandible, mesial to the root of the first molar. Controlled-release pellets locally delivered 0.1, 0.05, or 0.025 mg/day of PGE2. The right side of the mandible was used as a matched control for each animal. Six sham-treated animals were implanted with a placebo pellet. On days 7 and 19, animals were injected with the bone markers tetracycline and calcein, respectively. On day 21, animals were sacrificed and undecalcified tissues obtained for morphometrical analysis. Morphometrical measurements were analyzed by paired t test to determine differences between the matched samples and one-way ANOVA to compare the different treatment groups. A significant increase in alveolar bone area was observed in mandibles treated with 0.1 and 0.05 mg/day when compared with matched controls and the placebo group. This was accompanied by a significant increase in alveolar bone height and width. The proportions of double-labeled surface (dLS), the mineral apposition rate (MAR), and bone formation rate (BFR) were significantly increased in mandibles treated with the two higher doses of PGE2. The proportion of resorptive surface (RS) was significantly reduced in these two groups. It is concluded that PGE2 induces alveolar bone formation in the mandible when locally delivered at a dose of 0.1 or 0.05 mg/day for 20 days.

Incisor disocclusion in rats affects mandibular condylar cartilage at the cellular level.

UNLABELLED: The effect of altered occlusion on the mandibular condylar cartilage remains unclear. OBJECTIVE: This study investigated the effect of unilateral incisor disocclusion on cartilage thickness, on mitotic activity and on chondrocytes maturation and differentiation in the mandibular condylar cartilage of rats. DESIGN: The upper and lower left incisors were trimmed 2mm every second day in five rats. In other five rats, the incisor occlusion was not altered. Condylar tissues from both sides of each mandible were processed and stained for Herovici's stain and immunohistochemistry for bromodeoxyuridine (BrdU), transforming growth factor-beta1 (TGF-beta1), alkaline phosphatase (ALP) and osteocalcin (OCN). Measurements of cartilage thickness and the numbers of immunopositive cells for each antibody were analysed by one-way analysis of variance (ANOVA). RESULTS: No significant differences were observed in cartilage thickness after 7 days of unilateral incisor disocclusion. However, the numbers of immunopositive cells for BrdU as a marker of DNA synthesising cells, TGF-beta1 as a marker of chondrocytes differentiation, and ALP and OCN as markers of chondrocytes maturation, were significant higher in the cartilage cells on both sides when incisor occlusion was unilaterally altered. Interestingly, alkaline phosphatase was highly expressed on the condylar side of incisor disocclusion, whereas osteocalcin was highly expressed on the side opposite to the incisor disocclusion. CONCLUSIONS: It is demonstrated that after 7 days, unilateral incisor disocclusion affects the mandibular condylar cartilage at the cellular level by increasing the mitotic activity and by accelerating chondrocytes maturation. Chondrocytes maturation appears more accelerated on the side opposite to incisor disocclusion.

Dental characteristics of patients with Duchenne muscular dystrophy.

A comprehensive assessment of the dental characteristics of 23 patients with Duchenne muscular dystrophy (DMD) was carried out, based on dental records, oral examinations and dental models. Decreasing muscle function was associated with increased plaque and calculus accumulation, leading to gingival inflammation, but caries experience was low. Disturbances in tooth form, number and eruption of the second premolars were observed in 39% of patients. Anterior and posterior open bites were common, associated with lip incompetence, mouth breathing, macroglossia and tongue thrusting. Maxillary and mandibular arch breadths were significantly larger, on average, in the DMD group than in controls. Rather than a normal parabolic arch form, the dental arches in DMD patients tended to be hyperbolic, with the posterior teeth being displaced buccally, consistent with an imbalance between the lingual and facial musculature.

Pulpal heat changes with newly developed resin photopolymerisation systems.

Composite resin is a widely-used direct tooth coloured restorative material. Photoactivation of the polymerisation reaction can be achieved by visible blue light from a range of light sources, including halogen lamps, metal halide lamps, plasma arc lamps, and Light Emitting Diode (LED) lights. Concerns have been raised that curing lights may induce a temperature rise that could be detrimental to the vitality of the dental pulp during the act of photoactivation. The present study examined heat changes associated with standardised class V restorations on the buccal surface of extracted premolar teeth, using a curing time of 40 seconds. The independent effects of type of light source, resin shade, and remaining tooth thickness were assessed using a matrix experimental design. When a conventional halogen lamp, a metal halide lamp and two different LED lights were compared, it was found that both LED lamps elicited minimal thermal changes at the level of the dental pulp, whereas the halogen lamp induced greater changes, and the metal halide lamp caused the greatest thermal insult of all the light sources. These thermal changes were influenced by resin shade, with different patterns for LED versus halogen or halide sources. Thermal stress reduced as the remaining thickness of tooth structure between the pulp and the cavity floor increased. From these results, it is concluded that LED lights produce the least thermal insult during photopolymerisation of composite resins.

Growth hormone receptor and IGF-I receptor immunoreactivity during orthodontic tooth movement in the prednisolone-treated rat.

Bone remodeling during tooth movement is regulated by local and systemic factors. Two regulators of bone metabolism are growth hormone (GH) and insulin-like growth factor-I (IGF-I). Their effects are mediated via binding to GH receptor (GHR) and IGF-I receptor (IGF-IR) in target tissues. Corticosteroids may affect the activity of these growth factors. This study examined the effect of prednisolone on GHR and IGF-IR expression in dental tissues following orthodontic tooth movement. The corticosteroid-treated group (N = 6) was administered prednisolone (1 mg/kg) daily and the control group (N = 6) received equivalent volumes of saline. An orthodontic force (30 g) was applied to the maxillary first molar. Animals were sacrificed 12 days postappliance insertion. Sagittal sections of the first molar were stained for GHR and IGF-IR immunoreactivity. GHR and IGF-IR cell counts were elevated following appliance-treatment. Orthodontic tooth movement appeared to up-regulate GHR and IGF-IR immunoreactivity, but this up-regulation was reduced following prednisolone treatment. The suppression of GHR and IGF-I immunoreactivity in steroid-treated animals infers the mechanism whereby bone resorption and deposition, necessary for orthodontic tooth movement, may be inhibited by prednisolone. However, at 12 days postappliance insertion, no difference in orthodontic tooth movement was observed following low-dose prednisolone treatment.

Effect of a static magnetic field on orthodontic tooth movement in the rat.

Orthodontic tooth movement may be enhanced by the application of a magnetic field. Bone remodelling necessary for orthodontic tooth movement involves clastic cells, which are tartrate-resistant acid phosphatase (TRAP) positive and which may also be regulated by growth hormone (GH) via its receptor (GHR). The aim of this study was to determine the effect of a static magnetic field (SMF) on orthodontic tooth movement in the rat. Thirty-two male Wistar rats, 9 weeks old, were fitted with an orthodontic appliance directing a mesial force of 30 g on the left maxillary first molar. The appliance incorporated a weight (NM) or a magnet (M). The animals were killed at 1, 3, 7, or 14 days post-appliance insertion, and the maxillae processed to paraffin. Sagittal sections of the first molar were stained with haematoxylin and eosin (H&E), for TRAP activity or immunohistochemically for GHR. The percentage body weight loss/gain, magnetic flux density, tooth movement, width of the periodontal ligament (PDL), length of root resorption lacunae, and hyalinized zone were measured. TRAP and GHR-positive cells along the alveolar bone, root surface, and in the PDL space were counted. The incorporation of a SMF (100-170 Gauss) into an orthodontic appliance did not enhance tooth movement, nor greatly alter the histological appearance of the PDL during tooth movement. However significantly greater root resorption (P = 0.016), increased width of the PDL (P = 0.017) and greater TRAP activity (P = 0.001) were observed for group M at day 7 on the compression side. At day 14 no differences were observed between the appliance groups.

External apical root resorption following orthodontic treatment.

This study investigated the association of appliance type and tooth extraction with the incidence of external apical root resorption (EARR) of posterior teeth following orthodontic treatment. Pre- and posttreatment orthopantomograms were compared for 97 patients and a 4-grade ordinal scale used to measure EARR. The incidence of EARR was positively associated with tooth position (P < .001), appliance type (P = .038), and extractions (P = .001). This was observed in an overall analysis mutually adjusted for the effects of age at start of treatment, pretreatment overbite and overjet, use of headgear, tooth extraction, and type of appliance. The incidence of EARR was 2.30 times higher for Begg appliances compared with edgewise, and it was 3.72 times higher where extractions were performed.

Orthodontic tooth movement in the prednisolone-treated rat.

Adverse effects of corticosteroids on bone metabolism raise concerns as to whether steroid treatment may influence orthodontic movement. This study examined the effect of prednisolone on orthodontic movement using an established rat model. The corticosteroid treated group (N = 6) was administered prednisolone (1 mg/kg) daily, for a 12-day induction period; the control group (N = 6) received equivalent volumes of saline. On day 12, an orthodontic appliance was placed which exerted 30 g of mesial force to the maxillary first molar. Animals were sacrificed on day 24 and tooth movement was measured. Sagittal sections of the molars were stained with haematoxylin and eosin, and for tartrate-resistant acid phosphatase (TRAP) activity. While there were no significant differences in the magnitude of tooth movement between the 2 groups, steroid-treated rats displayed significantly less root resorption on the compression side and fewer TRAP-positive cells within the PDL space on the same side. This suggests steroid treatment suppressed clastic activity.

A histological study of the effect of growth hormone on odontogenesis in the Lewis dwarf rat.

The effect of growth hormone (GH) on the dentition has been described in children with pituitary dwarfism where teeth fail to form; those that do form tend to be reduced in size and the eruption potential is diminished. The aim here was to examine the effect of GH on odontogenesis via molar development in Lewis (control), dwarf (Dw) and Dw GH-treated (Dw+GH) rats aged 3, 6, 9, 12 and 15 days. Dw+GH animals received a twice-daily dose (65 microg/kg) of GH which commenced at 2 days of age. Animals were killed, mandibles removed, processed to embedding in paraffin, sectioned and stained for histological examination of molar morphology during development. Variations in enamel mineralization and root development were observed. In 6-day-old animals, enamel mineralization was delayed in Dw and Dw+GH animals. Root initiation was evident at 6 days of age in controls but was not observed until 9 days of age in Dw and Dw+GH animals. At 12 days of age, maturation of enamel in Dw and Dw+GH animals remained delayed. By 15 days of age no variation in tooth development was evident. These data indicate that enamel mineralization is affected by the level of circulating GH in the rat. A specific deficiency of GH did not appear to delay bone resorption prior to tooth emergence.

The effect of glucocorticosteroid treatment on dentine formation in the Lewis rat, a histological study.

Glucocorticosteroids are widely used in the treatment of chronic illnesses and have been reported to cause premature obliteration of the pulp space. During the active stages of dentinogenesis, odontoblasts are growth hormone receptor (GHr) positive. The aims of this study were to determine if the glucocorticosteroid, prednisone, affected the rate of dentine deposition and odontoblast expression of GHr in the rat molar. Following subcutaneous injection of 0.05 mg/kg, 1.0 mg/kg or 5.0 mg/kg prednisone for 20 days, immature and mature molars from rats aged 3 and 6 weeks respectively, were examined histologically. Distribution of GHr expression was determined immunohistochemically. No morphological differences were observed in molars from prednisone treated animals. Prednisone did not appear to enhance dentine deposition in immature molars but in mature molars significantly increased dentine deposition on the roof of the pulp chamber at a dosage of 5.0 mg/kg (p < 0.001). In all immature molars, odontoblasts and pulp cells expressed GHr immunoreactivity. In mature molars, odontoblasts and pulpal cells from controls did not show GHr immunoreactivity. However, odontoblasts and pulp cells were GHr immunoreactive in mature molars from animals treated with prednisone.

External apical root resorption of posterior teeth in asthmatics after orthodontic treatment.

External apical root resorption is an undesirable sequela of orthodontic treatment, resulting in loss of tooth structure from the root apex. It has been proposed that systemic factors, such as the inflammatory mediators produced in asthma, may enter the periodontal ligament and act synergistically to enhance root resorption. The aim of this study was to determine if asthmatic patients exhibited a higher incidence or severity of external apical root resorption compared with healthy (no medical conditions) patients after fixed orthodontic treatment. Records were obtained from patients treated with fixed appliances; 99 were healthy and 44 had asthma. Using OPGs (panoral films), posterior external apical root resorption was measured on all first and second premolars, mesiobuccal and distobuccal roots of the upper first molars, and mesial and distal roots of the lower first molars, giving 4 measurements per quadrant. A 4-grade ordinal scale was used to determine the degree of external apical root resorption. Combined tooth analysis (adjusted for treatment time, appliance, and extractions) showed that asthmatics had significantly more external apical root resorption of posterior teeth after treatment compared with the healthy group (P =.0194). Tooth-by-tooth analysis (adjusted for treatment time, appliance, extractions, headgear, overbite, overjet, sex, and age at start of treatment) found the upper first molars were most susceptible to external apical root resorption. Although the incidence of external apical root resorption was elevated in the asthma group, both asthmatics and healthy patients exhibited similar amounts of grade 2 (moderate) and grade 3 (severe) resorption.

Should cementoblasts express alkaline phosphatase activity? Preliminary study of rat cementoblasts in vitro.

BACKGROUND: A well-characterized cell culture model for cementoblasts is essential to understand the mechanisms of periodontal ligament (PDL) reattachment and regeneration. Whether cementoblasts express alkaline phosphatase (ALP) activity in vivo and in vitro remains to be determined. METHODS: Using a 2-step method of enzyme digestion/explant culture, osteoblasts, gingival/PDL fibroblasts, and cementoblasts were obtained from alveolar bone, gingiva, and the root surface of rat first molars and cultured. Initially, bone sialoprotein (BSP) was immunolocalized on tissue sections of periodontium and on cultured cells to distinguish mineral-forming cells from fibroblasts. Proteins were extracted from these cells to assess ALP activity by using an enzyme assay. RNA was extracted from the same cell source to detect ALP mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Cultured PDL/gingival fibroblasts were spindle shaped. Osteoblasts were irregularly shaped, and cell clusters/nodules were observed as they approached confluence. The cementoblasts manifested a polygonal shape and had two morphotypes: osteoblast-like and cuboidal or stratified. BSP was localized within the mineralized tissues and in osteoblasts and cementoblasts in culture and in tissue sections. The highest level of ALP activity was found in osteoblasts, a moderate level in PDL fibroblasts, and the lowest level in gingival fibroblasts. The cementoblasts lacked ALP activity, and this was reflected by a very weak signal (or no signal at all) for ALP mRNA in the cementoblasts. CONCLUSIONS: These studies indicate that cells consistent with a cementoblast-like phenotype may be successfully cultured, and that they lack ALP activity.

Insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) immunoreactivity in normal and osteopetrotic (toothless, tl/tl) rat tibia.

Insulin-like growth factor-I (IGF-I) plays a major role in regulating cell growth. This study examined the immunohistochemical distribution of IGF-I and IGF-I receptor (IGF-IR) in tibias from normal and osteopetrotic (toothless, tl/tl) rats, following treatment with colony stimulating factor-1 (CSF-1). In normal rats, immunoreactivity for IGF-I and IGF-IR was detected in cells of the articular and epiphyseal cartilage, secondary ossification centres, zones of resting and proliferating chondrocytes and bone marrow. Bone marrow cells immunoreactive for IGF-I and IGF-IR were significantly reduced in the tl/tl rat (p < 0.001) compared with normal animals. Treatment of tl/tl rats with CSF-1 increased immunoreactivity for IGF-I and IGF-IR in bone marrow cells as well as the number of TRAP positive osteoclasts. This increase was the result of recruitment of a range of hematopoietic cell types, including eosinophils, polymorphs and a substantial number of monocyte-like cells demonstrating strong immunoreactivity to IGF-I/IGF-IR. The differences in relative immunoreactivity for IGF-I/IGF-IR by bone marrow cells in untreated and CSF-1-treated tl/tl rats indicate a CSF-1-dependent recruitment of cells bearing surface IGF-IRs which may be mediated by an increase in local or systemic IGF-I.

Expression of transforming growth factor-beta receptors types II and III within various cells in the rat periodontium.

This study reports the immunohistochemical localization of TGF-beta receptor type II (T beta R-II) and type III (T beta R-III) in cells of the forming periodontal ligament (PDL) in rat first molar roots. Mandibular periodontium was obtained from 3, 6 and 12-wk-old rats. This represented tissue from the initial, pre-mature and post-mature stages of root and periodontal development, respectively. Mandibular bone chips and molar roots were used to isolate osteoblasts, fibroblasts and cementoblasts. Cells were obtained using a 2-step trypsinization and explant technique, and cultured in Dulbecco's modification of Eagle's medium (DMEM) under routine cell culture conditions. Cells were cultured on coverslips for the purpose of detecting TGF-beta receptors, and compared with whole tissue sections using the same detection method. Cells which stained positively for T beta R-II and T beta R-III on both paraffin sections and cultured cell slides were counted. Both receptors were expressed in the various periodontal tissue compartments. PDL fibroblasts, cementoblasts and osteoblasts were stained positively for T beta R-II and T beta R-III. Endothelial cells were noted to be positive for T beta R-II only. T beta R-II was more widely distributed in cells than T beta R-III, but T beta R-III was extensively localized in the extracellular matrix. Both receptors were expressed on the cell membrane and also localized in the cytoplasm. The findings for paraffin sections were consistent with the immunohistochemical staining of cultured cells. The percentage of cells which stained positively for T beta R-II was greater (approximately 85%) than that for T beta R-III (approximately 60%) in all major types of the PDL cells on both paraffin sections and cultured cell slides. Extensive location of TGF-beta receptors in both cells and extracellular matrix suggests that several binding sites are available for TGF-beta s to interact with target cells during development and following maturation of the periodontium.

Expression of transforming growth factor-beta 1 (TGF-beta1) in the developing periodontium of rats.

Transforming growth factor-beta 1 (TGF-beta1) has been reported to be expressed within several tissue compartments of developing molar crowns and therefore is implicated in tooth development. Additionally, TGF-beta1 may also play a crucial role in tissue repair and regeneration. The aim of this study was to determine the distribution of TGF-beta1 in the developing periodontal attachment apparatus (cementum, periodontal ligament, and alveolar bone) in Lewis rats. Animals aged 3, 6, and 12 wks were killed, their mandibles removed, fixed, demineralized, and processed in paraffin. The localization of TGF-beta1 in tissues was detected by polyclonal goat antibodies against human TGF-beta1 by means of immunoperoxidase techniques. TGF-beta1 messenger RNA was detected by in situ hybridization with a cocktail oligonucleotide probe. Cell counts were determined for analysis of the percentage of cells stained positive for TGF-beta1. Results revealed that TGF-beta1 was expressed in the developing alveolar bone, periodontal ligament, and cementum at all stages of tissue development studied. Staining was stronger at sites of cementum and alveolar bone compared with the periodontal ligament. Intensity of the positive staining, based on 3 grades, indicated a similarity between the tissues obtained from different ages, but varied between several cell types. Cementoblasts and osteoblasts stained more strongly than fibroblasts. Large numbers (approximately 90%) of the osteocytes in developing bone expressed TGF-beta1; however, in mature bone, fewer osteocytes stained for TGF-beta1. The percentages of positively stained cementoblasts, osteoblasts, and fibroblasts in the periodontal space were greater at the apical portion than at the cervical portion of the root. TGF-beta1 mRNA was expressed in osteoblasts, some bone marrow cells, cementoblasts, and fibroblasts. This study indicates that TGF-beta1 may play an important role in the modulation of tissue formation and development of the periodontium.

Preeruptive intracoronal radiolucencies: review and report of case.

Intracoronal radiolucencies in unerupted teeth are an uncommon radiographic finding; but their early detection and classification allow the most appropriate management protocol to be developed. Early separation of lesions into those that are developmental and remain static and those that are reactive and aggressive is necessary for a controlled outcome. The current paper reviews possible formative mechanisms and describes a case of severe intracoronal resorption resulting in loss of the tooth.