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.

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.

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.

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.

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.

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.

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.

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.

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.