Biological Evaluation of Oral Care Products Using 3D Tissue-Engineered In Vitro Models of Plaque-Induced Gingivitis.

BACKGROUND: The aim of this study was to investigate and visualize the anti-inflammatory and anti-bacterial effects of different oral care products using an infected and inflamed 3D tissue-engineered gingival mucosal model. METHODS: A 3D full-thickness oral mucosal model was engineered inside tissue culture inserts using collagen hydrogels populated with human gingival fibroblasts and THP-1 monocytes and layered with oral epithelial cell lines. Oral saliva bacteria were cultured and added to the surface of the models and inflammation was further simulated with lipopolysaccharide (LPS) of Escherichia coli. The 3D models were exposed to three different types of toothpastes, a chlorhexidine antiseptic mouthwash, different antibiotics, and a mechanical rinse with phosphate-buffered saline (PBS) prior to biological evaluation using the PrestoBlue tissue viability assay, histology, optical coherence tomography (OCT), confocal microscopy, and measurement of the release of the inflammatory markers IL-1ß, IL-6, and IL-8 with ELISA. RESULTS: Multiple-endpoint analyses of the infected oral mucosal models treated with different anti-bacterial agents showed consistent outcomes in terms of tissue viability, histology, OCT, and confocal microscopy findings. In terms of anti-inflammatory testings, the positive control group showed the highest level of inflammation compared with all other groups. Depending on the anti-bacterial and anti-inflammatory potential of the test groups, different levels of inflammation were observed in the test groups. CONCLUSIONS: The inflamed 3D oral mucosal model developed in this study has the potential to be used as a suitable in vitro model for testing the biocompatibility, anti-inflammatory, and anti-bacterial properties of oral care products including mouthwashes and toothpastes. The results of this study indicate that the chlorhexidine mouthwash has both anti-bacterial and cytotoxic effects on the 3D oral mucosal model. Hyaluronic-acid-containing toothpaste has significant anti-bacterial and anti-inflammatory effects on the 3D oral mucosal model.

Individual repeatability of avian migration phenology: A systematic review and meta-analysis.

Changes in phenology and distribution are being widely reported for many migratory species in response to shifting environmental conditions. Understanding these changes and the situations in which they occur can be aided by understanding consistent individual differences in phenology and distribution and the situations in which consistency varies in strength or detectability. Studies tracking the same individuals over consecutive years are increasingly reporting migratory timings to be a repeatable trait, suggesting that flexible individual responses to environmental conditions may contribute little to population-level changes in phenology and distribution. However, how this varies across species and sexes, across the annual cycle and in relation to study (tracking method, study design) and/or ecosystem characteristics is not yet clear. Here, we take advantage of the growing number of publications in movement ecology to perform a phylogenetic multilevel meta-analysis of repeatability estimates for avian migratory timings to investigate these questions. Of 2,433 reviewed studies, 54 contained suitable information for meta-analysis, resulting in 177 effect sizes from 47 species. Individual repeatability of avian migratory timings averaged 0.414 (95% confidence interval: 0.3-0.5) across landbirds, waterbirds and seabirds, suggesting consistent individual differences in migratory timings is a common feature of migratory systems. Timing of departure from the non-breeding grounds was more repeatable than timings of arrival at or departure from breeding grounds, suggesting that conditions encountered on migratory journeys and outcome of breeding attempts can influence individual variation. Population-level shifts in phenology could arise through individual timings changing with environmental conditions and/or through shifts in the numbers of individuals with different timings. Our findings suggest that, in addition to identifying the conditions associated with individual variation in phenology, exploring the causes of between-individual variation will be key in predicting future rates and directions of changes in migratory timings. We therefore encourage researchers to report the within- and between- individual variance components underpinning the reported repeatability estimates to aid interpretation of migration behaviour. In addition, the lack of studies in the tropics means that levels of repeatability in less strongly seasonal environments are not yet clear.

Individual consistency in migration strategies of a tropical seabird, the Round Island petrel.

BACKGROUND: In migratory species, the extent of within- and between-individual variation in migratory strategies can influence potential rates and directions of responses to environmental changes. Quantifying this variation requires tracking of many individuals on repeated migratory journeys. At temperate and higher latitudes, low levels of within-individual variation in migratory behaviours are common and may reflect repeated use of predictable resources in these seasonally-structured environments. However, variation in migratory behaviours in the tropics, where seasonal predictability of food resources can be weaker, remains largely unknown. METHODS: Round Island petrels (Pterodroma sp.) are tropical, pelagic seabirds that breed all year round and perform long-distance migrations. Using multi-year geolocator tracking data from 62 individuals between 2009 and 2018, we quantify levels of within- and between-individual variation in non-breeding distributions and timings. RESULTS: We found striking levels of between-individual variation in at-sea movements and timings, with non-breeding migrations to different areas occurring across much of the Indian Ocean and throughout the whole year. Despite this, repeat-tracking of individual petrels revealed remarkably high levels of spatial and temporal consistency in within-individual migratory behaviour, particularly for petrels that departed at similar times in different years and for those departing in the austral summer. However, while the same areas were used by individuals in different years, they were not necessarily used at the same times during the non-breeding period. CONCLUSIONS: Even in tropical systems with huge ranges of migratory routes and timings, our results suggest benefits of consistency in individual migratory behaviours. Identifying the factors that drive and maintain between-individual variation in migratory behaviour, and the consequences for breeding success and survival, will be key to understanding the consequences of environmental change across migratory ranges.

DNA diet profiles with high-resolution animal tracking data reveal levels of prey selection relative to habitat choice in a crepuscular insectivorous bird.

Given the global decline of many invertebrate food resources, it is fundamental to understand the dietary requirements of insectivores. We give new insights into the functional relationship between the spatial habitat use, food availability, and diet of a crepuscular aerial insectivore, the European Nightjar (Caprimulgus europaeus) by relating spatial use data with high-throughput sequencing (HTS) combined with DNA metabarcoding. Our study supports the predictions that nightjars collect a substantial part of their daily nourishment from foraging locations, sometimes at considerable distance from nesting sites. Lepidopterans comprise 65% of nightjars' food source. Nightjars tend to select larger species of Lepidoptera (>19 mm) which suggests that nightjars optimize the efficiency of foraging trips by selecting the most energetically favorable-larger-prey items. We anticipate that our findings may shed additional light on the interactions between invertebrate communities and higher trophic levels, which is required to understand the repercussions of changing food resources on individual- and population-level processes.

Implant Soft-Tissue Attachment Using 3D Oral Mucosal Models-A Pilot Study.

PURPOSE: The aim of this study was to investigate soft-tissue attachment to different metal, ceramic, and polymer implant surfaces using an inflamed, three-dimensional (3D), tissue-engineered, human oral mucosal model, as well as multiple-endpoint qualitative and quantitative biological approaches. METHODS: Normal human oral fibroblasts, OKF6/TERT-2 keratinocytes and THP-1 monocytes were cultured, and full-thickness, 3D oral mucosal models were engineered inside tissue culture inserts. Sand-blasted and acid-etched (SLA) and machined (M) titanium-zirconium alloy (TiZr; commercially known as Roxolid; Institut Straumann AG, Switzerland), ceramic (ZrO2), and polyether ether ketone (PEEK) rods (Ø 4 mm × 8 mm) were inserted into the center of tissue-engineered oral mucosa following a Ø 4mm punch biopsy. Inflammation was simulated with addition of the lipopolysaccharide (LPS) of Escherichia coli (E. coli) and tumor necrosis factor (TNF)-alpha to the culture medium. Implant soft-tissue attachment was assessed using histology, an implant pull-test with PrestoBlue assay, and scanning electron microscopy (SEM). RESULTS: Inflamed, full-thickness, 3D human oral mucosal models with inserted implants were successfully engineered and histologically characterized. The implant pull-test with PrestoBlue assay showed higher viability of the tissue that remained attached to the TiZr-SLA surface compared to the other test groups. This difference was statistically significant (p < 0.05). SEM analysis showed evidence of epithelial cell attachment on different implant surfaces. CONCLUSIONS: The inflamed, 3D, oral mucosal model has the potential to be used as a suitable in vitro test system for visualization and quantification of implant soft-tissue attachment. The results of our study indicate greater soft tissue attachment to TiZr-SLA compared to TiZr-M, ceramic, and PEEK surfaces.

Biomodification of a Class-V Restorative Material by Incorporation of Bioactive Agents.

Restoring subgingival class-V cavities successfully, demand special biological properties from a restorative material. This study aimed to assess the effects of incorporating bioactive materials to glass ionomer cement (GIC) on its mechanical and biological properties. Hydroxyapatite, chitosan, chondroitin sulphate, bioglass, gelatine and processed bovine dentin were incorporated into a GIC restorative material. Compressive strength, biaxial flexural strength (BFS), hardness, setting and working time measurements were investigated. Biocompatibility of the new materials was assessed using both monolayer cell cultures of normal oral fibroblasts (NOF) and TR146 keratinocytes, and a 3D-tissue engineered human oral mucosa model (3D-OMM) using presto-blue tissue viability assay and histological examination. Significant reduction in the compressive strength and BFS of gelatine-modified discs was observed, while chondroitin sulphate-modified discs had reduced BFS only (p value > 0.05). For hardness, working and setting times, only bioglass caused significant increase in the working time. NOF viability was significantly increased when exposed to GIC-modified with bovine dentine, bioglass and chitosan. Histological examination showed curling and growth of the epithelial layer toward the disc space, except for the GIC modified with gelatine. This study has highlighted the potential for clinical application of the modified GICs with hydroxyapatite, chitosan, bioglass and bovine dentine in subgingival class-V restorations.

Effects of electronic cigarette liquid on monolayer and 3D tissue-engineered models of human gingival mucosa.

Background: There is limited data available on potential biological effects of E-cigarettes on human oral tissues. The aim of this study was to evaluate the effects of E-cigarette liquid on the proliferation of normal and cancerous monolayer and 3D models of human oral mucosa and oral wound healing after short-term and medium-term exposure. Methods: Normal human oral fibroblasts (NOF), immortalized OKF6-TERET-2 human oral keratinocytes, and cancerous TR146 keratinocyte monolayer cultures and 3D tissue engineered oral mucosal models were exposed to different concentrations (0.1%, 1%, 5% and 10%) of E-cigarette liquid (12 mg/ml nicotine) for 1 hour daily for three days and for 7 days. Tissue viability was monitored using the PrestoBlue assay. Wounds were also produced in the middle surface of the monolayer systems vertically using a disposable cell scraper. The alterations in the cell morphology and wound healing were visualized using light microscopy and histological examination. Results: Statistical analysis showed medium-term exposure of TR146 keratinocytes to 5% and 10% E-liquid concentrations significantly increased the viability of the cancer cells compared to the negative control. Short-term exposure of NOFs to 10% E-liquid significantly reduced the cell viability, whereas medium-term exposure to all E-liquid concentrations significantly reduced the NOF cells' viability. OKF6 cells exhibited significantly lower viability following short-term and mediumterm exposure to all E-cigarette concentrations compared to the negative control. 3D oral mucosal model containing normal oral fibroblasts and keratinocytes showed significant reduction in tissue viability after exposure to 10% E-liquid, whereas medium-term exposure resulted in significantly lower viability in 5% and 10% concentration groups compared to the negative control. There was a statistically significant difference in wound healing times of both NOF and OKF6 cells after exposure to 1%, 5% and 10% E-cigarette liquid. Conclusion: Medium-term exposure to high concentrations of the E-cigarette liquid had cytotoxic effects on normal human oral fibroblasts and OKF6 keratinocytes, but a stimulatory cumulative effect on the growth of cancerous TR146 keratinocyte cells as assessed by the PrestoBlue assay and histological evaluation of 3D oral mucosal models. In addition, E-liquid exposure prolonged the wound healing of NOF and OKF6 oral mucosa cells.

Biologic assessment of antiseptic mouthwashes using a three-dimensional human oral mucosal model.

BACKGROUND: The biologic safety profile of oral health care products is often assumed on the basis of simplistic test models such as monolayer cell culture systems. We developed and characterized a tissue-engineered human oral mucosal model, which was proven to represent a potentially more informative and more clinically relevant alternative for the biologic assessment of mouthwashes. The aim of this study was to evaluate the biologic effects of alcohol-containing mouthwashes on an engineered human oral mucosal model. METHODS: Three-dimensional (3D) models were engineered by the air/liquid interface culture technique using human oral fibroblasts and keratinocytes. The models were exposed to phosphate buffered saline (negative control), triethylene glycol dimethacrylate (positive control), cola, and three types of alcohol-containing mouthwashes. The biologic response was recorded using basic histology; a cell proliferation assay; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tissue-viability assay; transmission electron microscopy (TEM) analysis; and the measurement of release of interleukin (IL)-1beta by enzyme-linked immunosorbent assay. RESULTS: Statistical analysis showed that there was no significant difference in tissue viability among the mouthwashes, cola, and negative control groups. However, exposure to the positive control significantly reduced the tissue viability and caused severe cytotoxic epithelial damage as confirmed by histology and TEM analysis. A significant increase of IL-1beta release was observed with the positive control and, to a lesser extent, with two of the tested mouthrinses. CONCLUSIONS: The 3D human oral mucosal model can be a suitable model for the biologic testing of mouthwashes. The alcohol-containing mouthwashes tested in this study do not cause significant cytotoxic damage and may slightly stimulate IL-1beta release.

Development of biodegradable electrospun scaffolds for dermal replacement.

Our objective is to develop a synthetic biodegradable replacement dermal substitute for tissue engineering of skin and oral mucosa. Our in vivo criteria were that candidate scaffolds should allow surrounding cells to migrate fully into the scaffolds, enabling vasculogenesis and remodelling without invoking a chronic inflammatory response. We examined a total of six experimental electrospun polymer scaffolds: (1) poly-l-lactide (PLLA); (2) PLLA+10% oligolactide; (3) PLLA+rhodamine and (4-6) three poly(d,l)-lactide-co-glycolide (PLGA) random multiblock copolymers, with decreasing lactide/glycolide mole fractions (85:15, 75:25 and 50:50). These were evaluated for degradation in vitro up to 108 days and in vivo in adult male Wistar rats from 4 weeks to 12 months. In vivo, all scaffolds permitted good cellular penetration, with no adverse inflammatory response outside the scaffold margin and with no capsule formation around the periphery. The breakdown rate for each scaffold in vitro versus in vivo was similar, and an increase in the ratio of polyglycolide to polylactide correlated with an increase in breakdown rate, as expected. Scaffolds of PLLA were stable in vivo even after 12 months whereas scaffolds fabricated from PLGA 85:15 and 75:25 revealed a 50% loss of mass after 4 and 3 months, respectively. In vitro PLGA 85:15 and 75:25 scaffolds were able to support keratinocyte, fibroblast and endothelial cell growth and extracellular matrix production, with evidence of new collagen production after 7 days. In conclusion, the data supports the development of PLGA 85:15 and 75:25 electrospun polymer scaffolds as potential degradable biomaterials for dermal replacement.