Biocompatibility and acid resistance of preformed crowns in children: an in vitro study.

PURPOSE: To investigate the in vitro biocompatibility of human gingival fibroblasts with preformed paediatric crowns and resistance to acid exposure at levels that simulate the oral environment. METHODS: This laboratory study investigated primary HGFs viability, metabolic activity, cytotoxicity, and apoptotic events on preformed metal crown discs, composite resin-coated wells, and monolithic zirconia fragments at 24, 48, and 72 h using the ApoTox-Glo Triplex assay. The PPCs were also immersed in 0.1% lactic acid, 0.2% phosphoric acid, or 10% citric acid for 7 days at 37 °C to reproduce conditions associated with dietary intake or gastric reflux. Samples were then subject to inductively coupled plasma optical emission spectrometry to quantitate the release of ions. RESULTS: The viability of HGFs on stainless steel and CR significantly declined at 48 and 72 h, representing potential cytotoxicity (p < 0.05). Cytotoxicity of HGFs was also higher for stainless steel and ZR compared to control (p < 0.05). PMCs and ZR crowns gave minimal ion release. Meanwhile, significant quantities of metallic ions, including copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn), were present in eluates from veneered-preformed metal crowns. CONCLUSION: As PPCs can be exposed to highly acidic environments for many years, thus the release of metallic ions from V-PMCs should form the further investigation in future studies.

Unfolded protein response is involved in the metabolic and apoptotic regulation of oral squamous cell carcinoma.

Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have been shown to be crucial in the pathogenesis and response to treatment in various cancers. However, such response has not been profiled in oral squamous cell carcinoma (OSCC), the most frequent form of cancer in the head and neck region. Cell lines derived from OSCC (SCC4, SCC15 and SCC25) and normal oral mucosa (OKF4, OKF6 and OKP7) were subjected to tunicamycin-induced ER stress (2.5 µg/mL for 24 h) after which the differential regulation of 84 key UPR/ER stress genes were assessed using Quantitative real-time reverse transcription polymerase chain reaction. The expression of the transcription factors SREBP1 and CREB3L3, and the activation of SREBP1, were examined using ELISA and a transcription factor assay. The expression of DDIT3 was immunohistochemically verified in OSCC tissue samples. SREBP1 and CREB3L3 were significantly up-regulated in OSCC with and without tunicamycin-induced ER stress. A significantly higher level of SREBP1 transcriptional activation was observed in OSCC. Apoptosis-associated genes (DDIT3, HTRA4 and HSPA1L) were also significantly up-regulated in OSCC upon ER stress induction. The findings demonstrated the involvement of UPR and ER stress in the pathogenesis of OSCC through the identification of apoptosis-associated genes (DDIT3, HSPA1L and HTRA4) and regulators of metabolism (SREBP1 and CREB3L3) as the key factors differentiating between normal and malignant oral keratinocytes.

Endocytosed silver nanoparticles degrade in lysosomes to form secondary nanoparticle structures during expression of autophagy genes in osteogenic cells.

Silver nanoparticles (AgNPs) are increasingly used in combination with biomaterials, such as bone grafts, to provide antimicrobial properties. Our research focused on the cytotoxic and intracellular uptake mechanism of AgNPs on osteogenic cells, and the affected gene expression of osteoblasts exposed to AgNPs. Osteoblast cells were found to be relatively resistant to AgNP exposure, compared to osteoclasts, with a higher IC50 and fewer adverse morphological features. AgNPs were endocytosed within lysosomes, which resulted in the secondary internal formation of curved AgO nano-chains assemblies within the cytosol. Furthermore, osteoblasts demonstrated an oxidative stress response, with autophagic cell death mechanisms, as indicated from qRT2-PCR analysis, with sustained upregulation of the protective gene Heme Oxygenase 1 reaching 86-fold by 48 hours (10 µg/mL). The internalization and fate of AgNPs in osteogenic cells, and the resulting impact on gene expression over time provide further understanding of the nanotoxicity mechanism of AgNPs.

The in vitro effect of VEGF receptor inhibition on primary alveolar osteoblast nodule formation.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a master regulator and is required for the effective coupling of angiogenesis and osteogenesis supporting both skeletal development and postnatal bone repair. A direct role for VEGF in intramembranous-derived osteoblast growth and differentiation is not clear. We investigated the expression of primary alveolar osteoblast VEGF receptors and the subsequent effects on mineralization and nodule formation in vitro following VEGFR inhibition. METHODS: Primary human alveolar osteoblasts (HAOBs) were cultured in the presence of VEGF receptor inhibitors, exogenous VEGF or the bisphosphonate, zoledronic acid. VEGF, VEGFR1 and VEGFR2 mRNA expression and nodule formation following 21 days of culture. VEGFR1 protein expression was examined using immunofluorescence after 48 h. RESULTS: The HAOBs expressed high levels of VEGF and VEGFR1 protein but VEGFR2 was not detected. The VEGFR1/2 inhibitors, ZM306416 and KRN633, lead to a dose-dependent decrease in mineralization. Treatment with zoledronic acid showed no difference in HAOB VEGF receptor expression. CONCLUSION: VEGF/VEGFR1 pathway appears to be important for intramembranous-derived osteoblast differentiation and maturation in vitro.

Efficacy and safety of alpha lipoic acid-capped silver nanoparticles for oral applications.

Silver nanoparticles (AgNPs) are widely studied for their broad-spectrum antimicrobial effects, and can be utilised readily in biomaterials, however the cellular safety of specific AgNP formulations should be profiled prior to clinical usage. This study determined the cytotoxic effect of small sized (6 nm) alpha lipoic acid capped-AgNPs on human gingival fibroblasts (HGF), as compared to ionic silver and clinical antiseptics. The metabolic pathway was investigated to determine the cellular effects on HGF cells. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) was established for a range of oral related bacteria. Results showed that cell viability decreased with increasing AgNP concentration, whereas lower concentrations of AgNPs, (≤5 µg ml-1) caused a significant increase in cell proliferation at 24 and 72 hour time points. The cytotoxicity profile of AgNPs exhibited significantly lower concentrations, relative to the dose of clinical efficacy, when compared to clinical antiseptics. Caspase 3/7 was not significantly altered when HGF cells were treated with 0.225 µg ml-1 AgNPs, indicating cell necrosis rather than apoptosis. Quantitative RT2-PCR detected an upregulation of genes associated with oxidative stress and the G2M cell cycle checkpoint at ≤4 hours, but expression levels returned to levels consistent with control cells at 24-96 hours. An MIC range of 2.5-12.5 µg ml-1 (min. Escherichia coli, Streptococcus mutans, S. mitis; max. Staphylococcus aureus) was determined across the bacterial species tested and an MBC range of 5-100 µg ml-1 (min. E. coli, max. S. mutans). The antimicrobial profile was similar to that of AgNO3 which suggested that the antimicrobial effect may be influenced by free Ag+ release. It was concluded that alpha lipoic acid capped AgNPs possess limited cytotoxic activity to HGF cells when compared to clinically utilised oral antiseptics, observed via the cellular recovery after initial AgNP treatment and a lack of cumulative cytotoxic effect, whilst maintaining a broad range antimicrobial effect of the AgNPs.

Alternative rib bone biopsy measurements to estimate changes in skeletal mineral reserves in cattle.

Rib bone biopsy samples are often used to estimate changes in skeletal mineral reserves in cattle but differences in sampling procedures and the bone measurements reported often make interpretation and comparisons among experiments difficult. 'Full-core' rib bone biopsy samples, which included the external cortical bone, internal cortical bone and trabecular bone (CBext, CBint and Trab, respectively), were obtained from cattle known to be in phosphorus (P) adequate (Padeq) or severely P-deficient (Pdefic) status. Experiments 1 and 2 examined growing steers and Experiment 3 mature breeder cows. The thickness of cortical bone, specific gravity (SG), and the amount and concentration of ash and P per unit fresh bone volume, differed among CBext, CBint and Trab bone. P concentration (mg/cc) was closely correlated with both SG and ash concentrations (pooled data, r=0.99). Thickness of external cortical bone (CBText) was correlated with full-core P concentration (FC-Pconc) (pooled data, r=0.87). However, an index, the amount of P in CBext per unit surface area of CBext (PSACB; mg P/mm2), was more closely correlated with the FC-Pconc (pooled data, FC-Pconc=37.0+146×PSACB; n=42, r=0.94, RSD=7.7). Results for measured or estimated FC-Pconc in 10 published studies with cattle in various physiological states and expected to be Padeq or in various degrees of Pdefic status were collated and the ranges of FC-Pconc indicative of P adequacy and P deficiency for various classes of cattle were evaluated. FC-Pconc was generally in the range 130 to 170 and 100 to 120 mg/cc fresh bone in Padeq mature cows and young growing cattle, respectively. In conclusion, the FC-Pconc could be estimated accurately from biopsy samples of CBext. This allows comparisons between studies where full-core or only CBext biopsy samples of rib bone have been obtained to estimate changes in the skeletal P status of cattle and facilitates evaluation of the P status of cattle.

FoxP3+ regulatory T cells, interleukin 17 and mast cells in chronic inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: T cells are known to play a pivotal role in periodontal disease; however, less is known about the T-helper subsets of regulatory T cells (Tregs) and Th17 cells. The aim of this study was to investigate the cell types expressing FoxP3 and interleukin (IL)-17A within periodontal disease tissues and to determine gene and protein expression profiles associated with periodontitis. MATERIAL AND METHODS: A total of 10 healthy/gingivitis and 10 chronic periodontitis tissues were investigated. Immunohistochemistry and immunofluorescence techniques were used to identify the FoxP3 and IL17-positive cells and to determine the cell types respectively. Gene expression was determined using semi-quantitative polymerase chain reaction array technology that allowed the analysis of 84 pathway-focused genes known to be associated with Tregs and Th17 cells. Transforming growth factor (TGF)-ß1, IL10 and IL17A protein levels were determined using enzyme-linked immunosorbent assay. RESULTS: Double immunofluorescence labeling revealed that all FoxP3+ cells were CD4+ , while IL17+ cells were neither CD4+ nor CD8+ but were tryptase+ , suggestive of mast cells. More FoxP3+ cells than IL17+ cells were found in all the tissues examined and overall there were few IL17+ cells. Statistically significant increases in gene expression were found for STAT5A, STAT3, SOCS1, TGFß1 and IL10 in the chronic periodontitis specimens predominantly infiltrated with B cells and plasma cells when compared with healthy/gingivitis specimens predominantly infiltrated with T cells. Protein analysis demonstrated higher levels of the TGFß1 and IL10 cytokines in periodontitis tissues and in B-cell and plasma cell predominant gingival tissues than in healthy/gingivitis tissues and T-cell predominant gingival tissues. IL17A gene and protein expression was not detected in any of the tissues. CONCLUSION: Based on the findings of this study, we suggest that the source of low levels of IL17A in periodontal tissues is mast cells not Th17 cells and that Tregs may have a more prominent role in the pathogenesis of periodontal disease than Th17 cells.

Attributes of Bio-Oss® and Moa-Bone® graft materials in a pilot study using the sheep maxillary sinus model.

BACKGROUND AND OBJECTIVE: The aim of this pilot study was to characterize surface morphology and to evaluate resorption and osseous healing of two deproteinated bovine bone graft materials after sinus grafting in a large animal model. MATERIAL AND METHODS: Surfaces of a novel particulate bovine bone graft, Moa-Bone® were compared with Bio-Oss® using scanning electron microscopy. Six sheep then had maxillary sinus grafting bilaterally, covered with BioGide® . Grafted maxillae were harvested after 4, 6 and 12 weeks. Healing was described for half of each site using resin-embedded ground sections. For the other half, paraffin-embedded sections were examined using tartrate resistant acid phosphatase staining for osteoclast activity, runt-related transcription factor2 immunohistochemistry for pre-osteoblasts and osteoblasts and proliferating cell nuclear antigen for proliferative cells. RESULTS: Moa-Bone® had a smoother, more porous fibrous structure with minimal globular particles compared with Bio-Oss® . After 4 weeks, woven bone formed on both grafts and the Moa-Bone® particles also showed signs of resorption. After 12 weeks, Moa-Bone® continued to be resorbed, however Bio-Oss® did not; both grafts were surrounded by maturing lamellar bone. Moa-Bone® was associated with earlier evidence of runt-related transcription factor 2-positive cells. Moa-Bone® but not Bio-Oss® was associated with strong tartrate resistant acid phosphatase-positive osteoclasts on the graft surface within resorption lacunae at both 4 and 6 weeks post-grafting. CONCLUSION: Both materials supported osseous healing and maturation without inflammation. Moa-Bone® showed marked osteoclast activity after 4 and 6 weeks and demonstrated positive attributes for grafting, if complete remodeling of the graft within the site is desired. Further optimization of Moa-Bone® for maxillofacial applications is warranted.

Effects of environmental tobacco smoke on the oral health of preschool children.

AIMS: This study investigated the association between the prevalence of oral health problems (caries, gingivitis, mucosal pigmentation and enamel defects in one to 5 year-old children exposed and not exposed to environmental tobacco smoke before and/or after birth. Exposure to environmental tobacco smoke (ETS) in childhood may have significant health effects. METHODS: A structured questionnaire was used to collect data on a child's current and previous illnesses, oral health behaviours, dietary habits, parental smoking behaviours and parents' dental history. The intraoral examination recorded dental caries (dmfs), enamel defects, gingival health, melanin pigmentation and soft tissue health. Stimulated saliva was collected. Total sIgA levels were quantified using indirect competitive ELISA with a SalimetricsTM kit. RESULTS: The 44 children (aged 15-69 months) recruited were divided into two groups: ETS and non-ETS (control). There were 22 children in each: 16 who were exposed to ETS during and after gestation were identified as the ETSB subgroup. Participants exposed to ETS were more likely to have had upper respiratory tract and middle ear infections during the neonatal period and had higher mean dmft, mean dmfs, mean percent of surfaces with demarcated opacities and mean GI than the non-ETS participants. The children exposed to ETS before and after birth had the highest occurrence of enamel opacities showed a higher risk for dental caries even though more children in this group used the recommended fluoride toothpaste (1000 ppm fluoride). Mothers who smoked either never breastfed their children or breastfed their children for less than the recommended period of 6 months. Children exposed to ETS were shown to have higher mean total sIgA (µg/ml) than the children in the control group. CONCLUSIONS: Associations between ETS exposure before and after gestation and oral health, including salivary changes in young children were shown in the present study. Dental health professionals should include a question about household smoking in children's dental histories, which would allow opportunities to discuss the impact of smoking on child oral health. Longitudinal oral health studies should include a history of maternal smoking during pregnancy and afterwards.

Contrasting Influences of Geographic Range and Distribution of Populations on Patterns of Genetic Diversity in Two Sympatric Pilbara Acacias.

The influence of geographic range on species persistence has long been of interest and there is a need for a better understanding of the genetic consequences for species with restricted distributions, particularly with the increasing rate of global species extinctions. However, the genetic effects of restricted range are often confounded by the impacts of population distribution. We compared chloroplast and nuclear genetic diversity and differentiation in two acacias, the restricted, patchily distributed Acacia atkinsiana and the widespread, semi-continuously distributed A. ancistrocarpa. Lower intra-population diversity and higher differentiation between populations were seen in A. atkinsiana compared to its widespread congener, A. ancistrocarpa. There was little evidence of geographical influences on population genetic structure in A. ancistrocarpa whereas A. atkinsiana exhibited nuclear genetic structure with isolation by distance, differentiation of near-coastal populations from those in the ranges, and differentiation of peripheral populations from those in the centre of the distribution. These results are consistent with expectations of the effect of geographic range and population distribution on genetic diversity, but indicate that distribution of populations rather than geographic range has influenced the observed genetic structure. The contrasting patterns observed here demonstrate that conservation approaches for species management and ecological restoration need to consider the distribution of populations in geographically restricted species.

Randomised comparison of the effectiveness of the laryngeal mask airway supreme, i-gel and current practice in the initial airway management of out of hospital cardiac arrest: a feasibility study.

BACKGROUND: The best initial approach to advanced airway management during out of hospital cardiac arrest (OHCA) is unknown. The traditional role of tracheal intubation has been challenged by the introduction of supraglottic airway devices (SGAs), but there is contradictory evidence from observational studies. We assessed the feasibility of a cluster-randomized trial to compare the i-gel SGA vs the laryngeal mask airway supreme (LMAS) vs current practice during OHCA. METHODS: We conducted a cluster-randomized trial in a single ambulance service in England, with individual paramedics as the unit of randomization. Consenting paramedics were randomized to use either the i-gel or the LMAS or usual practice for all patients with non-traumatic adult OHCA, that they attended over a 12-month period. The primary outcome was study feasibility, including paramedic and patient recruitment and protocol adherence. Secondary outcomes included survival to hospital discharge and 90 days. RESULTS: Of the 535 paramedics approached, 184 consented and 171 attended study training. Each paramedic attended between 0 and 11 patients (median 3; interquartile range 2-5). We recruited 615 patients at a constant rate, although the LMAS arm was suspended in the final two months following three adverse incidents. The study protocol was adhered to in 80% of patients. Patient characteristics were similar in the three study arms, and there were no differences in secondary outcomes. CONCLUSION: We have shown that a prospective trial of alternative airway management strategies in OHCA, cluster randomized by paramedic, is feasible. CLINICAL TRIAL REGISTRATION: Registered on the International Standard Randomised Controlled Trial Registry ( ISRCTN: 18528625).

Effects of zoledronic acid and geranylgeraniol on the cellular behaviour and gene expression of primary human alveolar osteoblasts.

INTRODUCTION: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious complication of bisphosphonate therapy. The mechanism underlying BRONJ pathogenesis is poorly understood. OBJECTIVES: To determine the effects of zoledronic acid (ZA) and geranylgeraniol (GGOH) on the mevalonate pathway (MVP) in osteoblasts generated from the human mandibular alveolar bone in terms of cell viability/proliferation, migration, apoptosis and gene expression. MATERIALS AND METHODS: Primary human osteoblasts (HOBs) isolated from the mandibular alveolar bone were phenotyped. HOBs were cultured with or without ZA and GGOH for up to 72 h. Cellular behaviour was examined using a CellTiter-Blue® viability assay, an Ibidi culture-insert migration assay, an Apo-ONE® Homogeneous Caspase-3/7 apoptosis assay and transmission electron microscopy (TEM). Quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR) was used to determine the simultaneous expression of 168 osteogenic and angiogenic genes modulated in the presence of ZA and GGOH. RESULTS: ZA decreased cell viability and migration and induced apoptosis in HOBs. TEM revealed signs of apoptosis in ZA-treated HOBs. However, the co-addition of GGOH ameliorated the effect of ZA and partially restored the cells to the control state. Twenty-eight genes in the osteogenic array and 27 genes in the angiogenic array were significantly regulated in the presence of ZA compared with those in the controls at one or more time points. CONCLUSION: The cytotoxic effect of ZA on HOBs and its reversal by the addition of GGOH suggests that the effect of ZA on HOBs is mediated via the MVP. CLINICAL RELEVANCE: The results suggest that GGOH could be used as a possible therapeutic/preventive strategy for BRONJ.

The bisphosphonate zoledronic acid regulates key angiogenesis-related genes in primary human gingival fibroblasts.

BACKGROUND: Osteonecrosis of the jaws is recognised as a serious complication for patients receiving bisphosphonates. The anti-angiogenic effects of bisphosphonates have been implicated in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). The purpose of this study was to determine the effects of zoledronic acid on cultured human gingival fibroblasts in relation to the modulation of genes associated with angiogenic regulation. METHODS: Primary cultures of fibroblasts were developed from gingival tissues excised during crown-lengthening surgery from three patients. Cells were cultured with and without 30µM zoledronic acid for 6, 12 and 24h and cellular proliferation and migration investigated using CellTiter-Blue and scratch wound assays, respectively. Gene expression was determined using semi-quantitative PCR array technology that allowed the analysis of 84 pathway-focused genes known to be important in the regulation of angiogenesis. RESULTS: Zoledronic acid increased the proliferation of the gingival fibroblasts in a dose dependent manner with 12 and 24h of exposure. Scratch wounding of the human gingival fibroblasts and treatment with increasing doses and time exposure to zoledronic acid (ZA) inhibited their migration. Statistically significant increases in gene expression were found for RHOB, VEGFA, CD55 and BMP2 (p≤0.05) in response to 30µM zoledronic acid. CCL2 and IL6 genes were significantly downregulated (p≤0.05). CONCLUSIONS: The regulation of the prenylated protein RHOB in this study was consistent with the known effects of zoledronic acid on the mevalonate pathway. The down regulation of CCL2 and IL6 and the upregulation of CD55 may be associated with suppression of inflammation. An increase in VEGFA and BMP2 gene expression suggests that fibroblasts respond to zoledronic acid by producing a proangiogenic environment.

Periodontopathogen levels following the use of an Er:YAG laser in the treatment of chronic periodontitis.

BACKGROUND: Inflammatory periodontal diseases are initiated by microbial biofilms. The reduction of the biofilm is important in the management of the disease. This study compares periodontopathogen levels following the treatment of chronic periodontitis using Er:YAG laser (ERL) debridement and mechanical scaling and root planing (SRP). METHODS: Using a split-mouth design, two quadrants were randomly allocated for treatment. Two hundred and fifty-two subgingival plaque samples were collected from 21 patients, before treatment (baseline) and at 6 and 12 weeks post-therapy. Multiplex qPCR was used to determine relative levels of Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythensis (Tf), and Aggregatibacter actinomycetemcomitans (Aa). RESULTS: Tf and Pg were significantly reduced post-treatment for both ERL and SRP. ERL treatment resulted in a reduction of Td at 12 weeks. Following SRP treatment Aa was significantly reduced at 12 weeks. No statistically significant difference was seen when treatments were compared at 6 and 12 weeks. CONCLUSIONS: A comparable reduction in the level of the four periodontal pathogens assayed was achieved with Er:YAG laser debridement and mechanical scaling and root planing.

Localization of RANK, RANKL and osteoprotegerin during healing of surgically created periodontal defects in sheep.

BACKGROUND AND OBJECTIVE: Modeling of periodontal bone regeneration in a large animal enables better examination of the spatial and temporal regulation of osteogenesis and the remodeling of the healing defect. RANK, RANKL and osteoprotegerin (OPG) are known to be important regulators of bone healing. The aim of this study was to create periodontal defects surgically in a large animal model and to examine bone regeneration and the expression of RANK, RANKL and OPG proteins in the defect site during bone regeneration. MATERIAL AND METHODS: Periodontal defects were made in the furcation of the second mandibular premolar of sheep. Wound healing was examined 6 h, and 1, 4 and 6 wk after surgery and in control tissue. The teeth and defect region were decalcified and paraffin embedded. Immunohistochemistry for RANK, RANKL and OPG was conducted. Osteoclasts were identified using TRAP staining. RESULTS: The defects were examined at different time points after surgery and by 6 wk the defect region had fully regenerated with new bone, albeit less dense than that in the unwounded controls. RANK-positive osteoclasts were present at the edge of the wound from week 1 and were found within the defect at week 6, corresponding to osteoclast activation and bone remodeling. RANKL staining increased from week 1 compared with unwounded tissue, and peaked at 4 and 6 wk, as the osteoblast numbers increased. At the same time, OPG immunostaining was high in controls and at week 6, suggesting that it may act to block RANKL and control the bone remodeling within the defect. CONCLUSION: Distinctive temporal and spatial expression patterns for RANK, RANKL and OPG proteins were observed during healing of surgically created periodontal wounds in a sheep model. The research identifies possible therapeutic approaches to periodontal bone repair via modulation of these members of the tumor necrosis factor family.

Zoledronic acid and geranylgeraniol regulate cellular behaviour and angiogenic gene expression in human gingival fibroblasts.

The mevalonate pathway (MVP) and the anti-angiogenic effect of bisphosphonates have been shown to play a role in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ). This study determined the effect of the bisphosphonate, zoledronic acid and the replenishment of the MVP by geranylgeraniol on human gingival fibroblasts. Cell viability, apoptosis, morphological analysis using transmission electron microscopy, and gene expression for vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B, epiregulin and interferon-alpha were conducted. Results showed cellular viability was decreased in the presence of zoledronic acid and the co-addition of zoledronic acid with geranylgeraniol restored cell viability to control levels. Caspase 3/7 was detected in zoledronic-acid-treated cells indicating apoptosis. Transmission electron microscopy revealed dilation of the rough endoplasmic reticulum with zoledronic acid and the appearance of multiple lipid-like vesicles following the addition of geranylgeraniol. Zoledronic acid significantly (P < 0.05, FR > ± 2) up-regulated vascular endothelial growth factor A, bone morphogenic protein 2, ras homologue gene family member B and epiregulin at one or more time points but not interferon-alpha. Addition of geranylgeraniol resulted in a reduction in the expression of all five genes compared with zoledronic-acid-treated human gingival fibroblasts. The study concluded geranylgeraniol partially reversed the effects of zoledronic acid in human gingival fibroblasts both at the cellular and genetic levels, suggesting the regulation of these genes is mediated via the mevalonate pathway.

Forkhead box P3-positive regulatory T-cells and interleukin 17-positive T-helper 17 cells in chronic inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: The role of two recently identified and closely related T-helper cell subsets - regulatory T-cells [Tregs; forkhead box P3-positive (FOXP3(+) )] and Th17 cells [interleukin-17-positive (IL-17(+) )] - in periodontal disease is yet to be determined. Tregs are essential in maintaining peripheral tolerance and regulating the immune response. Th17 cells play a critical role in several autoimmune diseases, inflammation and host defence. The aim of this study was to determine the presence of FOXP3(+) Tregs and IL-17(+) cells, and their possible spatial interaction, in diseased periodontal tissues. MATERIAL AND METHODS: Twenty-nine archival tissues with nonspecific gingival inflammation were grouped based on the intensity (minimally or intensely inflamed) and nature (T-cell predominant or B- and plasma-cell predominant) of the inflammatory infiltrate. Using double-labelling immunohistochemistry, the concomitant presence of FOXP3(+) and IL-17(+) cells was determined and their spatial relationship was established. In addition, the proportions of FOXP3(+) and IL-17(+) cells were compared between the groups. RESULTS: Of the 29 gingival specimens investigated, 17 were intensely inflamed (≥ 1000 inflammatory cells per 0.12 mm(2) ) and 12 were minimally inflamed (≤ 600 cells per 0.12 mm(2) ). Based on the percentage of CD19(+) B-cells and plasma cells collectively and CD3(+) T-cells, gingival tissues were also grouped into B- and plasma-cell-predominant gingival tissues (n = 21; 50.7% total B- and plasma cells vs. 19.1% T cells; p < 0.001) and T-cell-predominant gingival tissues (n = 8; 61.0% T-cells vs. 15.2% B- and plasma cells; p = 0.007). More FOXP3(+) cells than IL-17(+) cells were observed in all archival gingival tissues examined. A trend towards an increased number of FOXP3(+) cells was observed for intensely inflamed gingival tissues (6.7%) and for B- and plasma-cell-predominant tissues (6.4%) compared with minimally inflamed gingival tissues (4.6%) and T-cell-predominant gingival tissues (4.5%). However, no statistically significant difference in the mean percentage of FOXP3(+) cells between the groups was observed. Interestingly, FOXP3(+) cells were significantly correlated with the B- and plasma-cell/T-cell ratio in B- and plasma-cell-predominant tissues (r = 0.713, p < 0.001). Overall, there were very few IL-17(+) cells (< 1%). All IL-17(+) cells identified in this study had an ovoid/plasmacytoid morphology and were larger in size compared with adjacent inflammatory cells. IL-17(+) and FOXP3(+) cells were not adjacent to each other in any of the areas examined, suggesting that FOXP3(+) Tregs do not directly interact with IL-17(+) cells in diseased gingival tissues. IL-17(+) /FOXP3(+) cells were not detected in the tissues examined. CONCLUSION: These results show that FOXP3(+) cells are more prominent than IL-17(+) cells in periodontal disease processes, which may suggest a predominant role for FOXP3(+) cells in periodontal disease. Further studies are required to characterize these cells more precisely and to understand, in more detail, their roles in the pathophysiology of periodontal disease.

Genetic connectivity and diversity in inselberg populations of Acacia woodmaniorum, a rare endemic of the Yilgarn Craton banded iron formations.

Historically rare plant species with disjunct population distributions and small population sizes might be expected to show significant genetic structure and low levels of genetic diversity because of the effects of inbreeding and genetic drift. Across the globe, terrestrial inselbergs are habitat for rich, often rare and endemic flora and are valuable systems for investigating evolutionary processes that shape patterns of genetic structure and levels of genetic diversity at the landscape scale. We assessed genetic structure and levels of genetic diversity across the range of the historically rare inselberg endemic Acacia woodmaniorum. Phylogeographic and genetic structure indicates that connectivity is not sufficient to produce a panmictic population across the limited geographic range of the species. However, historical levels of gene flow are sufficient to maintain a high degree of adaptive connectivity across the landscape. Genetic diversity indicates gene flow is sufficient to largely counteract any negative genetic effects of inbreeding and random genetic drift in even the most disjunct or smallest populations. Phylogeographic and genetic structure, a signal of isolation by distance and a lack of evidence of recent genetic bottlenecks suggest long-term stability of contemporary population distributions and population sizes. There is some evidence that genetic connectivity among disjunct outcrops may be facilitated by the occasional long distance dispersal of Acacia polyads carried by insect pollinators moved by prevailing winds.

Evaluating the influence of different aspects of habitat fragmentation on mating patterns and pollen dispersal in the bird-pollinated Banksia sphaerocarpa var. caesia.

Habitat fragmentation can significantly affect mating and pollen dispersal patterns in plant populations, although the differential effects of the various aspects of fragmentation are poorly understood. In this study, we used eight microsatellite loci to investigate the effect of fragmentation on the mating system and pollen dispersal within one large and eight small population remnants of Banksia sphaerocarpa var. caesia, a bird-pollinated shrub in the southern agricultural region of Western Australia. The large population had a much larger neighbourhood size and lower selfing rate, maternal pollen pool differentiation and within-plot mean pollen dispersal distance than the small populations. Outcrossing was consistently high and ranged from 85.7% ± 2.6 to 98.5% ± 0.9, and mating patterns suggested nearest-neighbour pollination. Pollen immigration into small populations ranged from 2.8% ± 1.8 to 16.5% ± 3.2. Using the small populations, we tested for correlations between various fragmentation variables and mating system and pollen dispersal parameters. We found significant negative linear relationships between population isolation and outcrossing rate; population shape and neighbourhood size; and conspecific density and mean pollen dispersal distance. There were significant positive linear relationships between population shape and pollen pool differentiation and between population size and number of different fathers per seed crop. Our results suggest that birds may use a series of fragmented populations as a vegetation corridor while foraging across the landscape and that population connectivity is a critical determinant of pollinator visitation. Our results also suggest that the effect of a linear population shape on the mating system and pollen dispersal is routinely underestimated.

Attitudes of New Zealand dentists, dental specialists and dental students towards employing dual-trained Oral Health graduates.

AIMS: To determine the attitudes of New Zealand dentists and dental specialists towards employing dual-trained Oral Health (dental therapy/dental hygiene) graduates, their knowledge of the scopes of practice and practising requirements for Oral Health (OH) graduates, and the barriers to employment of these graduates. MATERIALS AND METHODS: A postal questionnaire was sent to 600 dentists randomly selected from the Dental Council of New Zealand register, as well as all dental specialists on the register. All fifth-year dental students in 2008 were also surveyed. RESULTS: The response rates for the questionnaires were 66.8% for dentists, 64.5% for dental specialists (specialists) and 72.9% for dental students. Knowledge of the scopes of practice and practising requirements for OH graduates was limited in some areas. Fifty-nine percent of private dental practitioners (PDP dentists) and 53% of specialists would consider employing an OH graduate. The main reason given for not employing an OH graduate was insufficient physical space in the practice. CONCLUSION: New Zealand dentists and dental specialists were receptive to employing OH graduates. Knowledge of the OH scopes of practice and practising requirements is likely to improve as more OH students graduate and start work. The OH graduates have the potential to make a valuable contribution to the dental team.