A digital method for estimating orthodontic treatment need

C Cai, S Olliver, L Mei, J Broadbent

Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin.

Dental malocclusions refer to the misalignment of teeth and may range from trivial to debilitating. The Dental Aesthetic Index (DAI) is commonly used to identify individuals with very severe malocclusions who may qualify for publicly funded care, but estimation of DAI scores may vary between referring dentists. The 3Shape Trios intra-oral scanner generates accurate three-dimensional digital images of the oral cavity, enabling the calculation of DAI digitally. However, no published reports have described such a method. We aimed to determine whether a modern digital imaging technique may enable more reliable estimation of DAI scores and allow its use in the Dunedin Multidisciplinary Health and Development Study (DMHDS).

World Health Organization criteria were used for estimating clinical DAI scores and were adapted to our digital method where necessary. DAI was measured both clinically and digitally by two examiners on a sample of 16 individuals presented with varying occlusal traits: a total of four times for eight participants and eight times for the remaining eight participants. After examiner calibration was complete, DAI was then collected for DMHDS participants.

DAI scores obtained clinically ranged from 19 to 37 (mean 27.2, sd 5.0), but test-retest scores varied by a mean of 1.0 point (sd 1.1). DAI scores obtained digitally ranged from 19 to 35 (mean 26.8, sd 4.6) and test-retest scores varied by a mean of only 0.1 points (sd 1.1). Both individual and average intra-class correlations based on consistency agreement and two-way mixed effects model were high, at >0.95 for all measurements of intra- and inter-examiner reliability. DAI scores in the Dunedin Study were strongly associated with self-perceived dental appearance, reaffirming the validity of the index.

This study demonstrating digital measurement of DAI scores allows reliable, rapid and accurate estimation of orthodontic treatment need outside the clinical setting.

Supported by a grant from the Auckland Dental Association Summer Scholarship.

The role of macrophages in allogenic skin graft rejection in murine models

T McBride,1,2 Z Lateef,1 L Wise1

1Department of Pharmacology and Toxicology; 2Department of Microbiology and Immunology, School of Biomedical Sciences, University of Otago, Dunedin.

An allograft is transplantation of skin from a genetically unrelated donor to a victim with a large area of uncovered trauma. While an allograft can restore organ function, rejection is a frequent problem due to the cellular destruction instigated by immune cells, such as macrophages. Immunosuppressants delay but do not prevent rejection, suggesting certain immune cells may be critical to graft integration. Macrophages can be categorised into two generalised subsets: M1, which are pro-inflammatory and M2, which promote tissue repair. We aimed to define the role of M1 and M2 macrophages in allograft rejection and integration in a murine model.

C57BL/6 and BALB/c mice were used as they have distinct immune responses. Full-thickness skin punch biopsies were taken from a donor C57BL/6 mouse, transplanted into full-thickness wounds on BALB/c mice and vice versa. Three C57BL/6 and four BALB/c mice received grafts. Mice were euthanised and the wound harvested on the day of estimated 50% rejection, C57BL/6 mice were euthanised one day prior to BALB/c mice. Immune cells within the grafts were identified using flow cytometry and immunofluorescent histochemistry; statistical significance was assessed with an unpaired t-test.

Two C57BL/6 and three BALB/c mice retained grafts. When the graft remained, tissue showed greater viability and integration in wounds of BALB/c mice. C57BL/6 and BALB/c mice had higher numbers of macrophages at the wound site, compared to controls; indicating an immune response. C57BL/6 mice had higher frequencies of M1 macrophages within wounds with grafts (mean ± SEM, C57BL/6 75.94±11.12%, BALB/c 22.85±6.74%, P<0.05); BALB/c mice displayed higher frequencies of M2 macrophages (BALB/c 10.67±1.61%, C57BL/6 5.35±2.24%, P<0.05).

This study implicates M1 cells with a role in allograft skin rejection and M2 cells in skin graft integration, both are therefore a possible drug target.

Supported by a School of Biomedical Sciences Summer Research Scholarship.

Aortic size index to predict risk in coronary artery disease patients

AW Blake-Barlow,1 S Coffey,1 GT Jones2

1Department of Medicine; 2Department of Surgical Sciences, Dunedin School of Medicine, University of Otago, Dunedin.

Cardiovascular disease is one of the leading causes of death and disability worldwide. Current risk stratification and classification of coronary artery disease (CAD) can be performed using coronary angiography. However, significant risk is not accounted for with this approach. We investigated the use of aortic size index (ASI, abdominal aortic diameter divided by body surface area) as a means of more accurate prognostication.

The cohort consisted of a consecutive series of patients undergoing coronary angiography due to suspicion of stable or unstable CAD. ASI was calculated based on an abdominal ultrasound. Standard demographic variables were obtained, and patients were stratified based on severity of their disease (obstructive, non-obstructive or no luminal disease). Adverse events were defined as myocardial infarction, hospitalisation due to unstable angina, unplanned revascularisation procedure or death due to any cause. ASI was analysed as a continuous measure as a predictor of events using logistic regression, and then as a categorical measure (extreme ASI, referring to largest and smallest ASI groups based on predefined cut points (lower <0.835cm/m2 and upper >1.2cm/m2) using Cox proportional hazards regression.

Data from 867 patients were analysed (mean age 68 years, 65% male). ASI showed a U shape relationship with outcomes where extremes of ASI had a higher risk of a subsequent event. Survival analysis revealed a 40% increase in event rate for those with ASI extremes (hazard ratio 1.40, 95% confidence interval 1.07–1.84, P=0.02), which remained significant when adjusted for age and sex. However, no significant relationship was seen with ASI when history of diabetes and overall CAD classification were included in the model (hazard ratio 1.21, 95% CI 0.92–1.59, P=0.18).

In conclusion, ASI is predictive of adverse events in patients undergoing coronary angiography, but does not add significant information over traditional predictors of adverse outcome.

Supported by a Division of Health Sciences Summer Student Research Scholarship.

Assessing the efficacy of a novel adeno-associated viral capsid in targeting the brain

SN Mathiesen,1,2 SM Ohline,1 HE Wicky,2 MYI Cheong,2 WC Abraham,1 SM Hughes2

1Department of Psychology; 2Department of Biochemistry, School of Biomedical Sciences , Brain Health Research Centre, University of Otago, Dunedin.

One barrier to the treatment of Alzheimer’s disease is a lack of effective methods to deliver therapeutics to the brain, particularly by non-invasive routes. Adeno-associated viral (AAV) vectors used for gene therapy may provide a solution as some serotypes can cross the blood brain barrier, allowing for minimally non-invasive administration. A novel AAV capsid, AAV-PHP.eB, was recently reported to produce high transgene expression in mouse brain after intravenous administration. The present study compared the efficiency of two AAV vectors in targeting the brain after intravenous administration: AAV-PHP.eB, and a similar widely used viral vector, AAV9.

C75Bl/6 mice were administered either AAV-PHP.eB (n=5) or AAV9 (n=4) carrying the transgene for green fluorescent protein (GFP) via tail vein injection (100µL). Four weeks after vector administration, brain sections were immunolabelled for GFP and cell-type specific markers. Liver sections were also examined for GFP expression. The small number of cells expressing GFP in both vector groups meant results were limited to qualitative observations.

Like AAV9, AAV-PHP.eB produced GFP expression in both neurons and glia. However, GFP expression in the brain produced by AAV-PHP.eB was not as high as reported. Despite transduction being sparse, AAV-PHP.eB was more effective than AAV9 at transducing cells across the whole brain. AAV-PHP.eB also produced much lower GFP expression in the liver than AAV9, which suggests that AAV-PHP.eB has high tropism for brain cells. The low liver transduction may mean more of the vector is available to reach the brain.

The higher efficacy of AAV-PHP.eB compared to AAV9 at transducing brain cells following intravenous delivery is encouraging when considering translation of this approach to humans. After additional work to evaluate the effectiveness of AAV-PHP.eB, it is planned to test this vector in future preclinical studies involving animal models of Alzheimer’s disease.

Supported by a Neurological Foundation of New Zealand Summer Student Scholarship.

Nanosilver as an antimicrobial for grafting materials and its cytotoxicity on human gingival fibroblasts in vitro

A Jude,1 C Gee,1 G Cotton,1 W Duncan,1 S Safii,2 D Coates1

1Sir John Walsh Research Institute, Faculty of Dentistry, University of Otago, Dunedin; 2Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia.

Moa Bone® is a deproteinated bovine bone product which increases clinically important bone regeneration in dental procedures. However, graft sites are often associated with infections. Silver nanoparticles (AgNP) represent a promising antimicrobial, yet limited information is available on their safety profile.

We investigated the cytotoxicity of AgNP on human gingival fibroblasts (HGF; N=3) using both in vitro cell viability and caspase 3/7 apoptosis assays. Chlorhexidine (CHX), silver nitrate (AgNO3) and silver diamine fluoride (SDF) were tested as widely accepted licensed clinical controls. Data was calculated as a mean ± SD of pairwise comparisons where controls of untreated cells were set to 100%. Paired t-tests were used for comparisons and IC50 regression analysis conducted in GraphPad PRISM7.

AgNP (22.5μg/mL for 4h) resulted in significant cytotoxicity to HGF with only 8.42±14.58% (P=0.0083) of cells being viable as compared to control (100%). However, no significant cytotoxic effect was seen at doses below 5μg/mL. CHX is used clinically at 0.2% and was cytotoxic at 0.02% after 4h (9.80±10.96%, P=0.0049). AgNO3 was cytotoxic at 50μg/mL after 4h (0.43±0.75%, P=0.00002). SDF is used clinically at 334,000μg/mL and was cytotoxic at 3.4μg/mL after 4h (7.26±2.60%, P=0.0021). AgNP had an IC50 of 10.2μg/mL at 4h and 10.6μg/mL at 96h, indicating minimal cumulative effect. Additionally, AgNP showed a statistically significant anti-apoptotic effect at 22.5μg/mL (39.78±5.74%, P=0.0031) and 50μg/mL (62.93±3.56%, P=0.002).

The cytotoxic effect of AgNP on HGF was similar to or less than that of CHX, AgNO3 and SDF and it was anti-apoptotic. Overall, AgNP showed a promising cytotoxicity profile which warrants its investigation as an adjunct to Moa Bone®.

Supported by a University of Otago Summer Studentship from the Otago Medical Research Foundation.

Elucidating the effect of prenatal androgen excess on male reproductive function and metabolism

S Holland,1 M Prescott,2 E Desroziers,2 R Campbell1

1Department of Physiology and Center for Neuroendocrinology, School of Biomedical Sciences, University of Otago, Dunedin.

Elevated maternal androgen levels are associated with the development of polycystic ovary syndrome (PCOS, a prevalent metabolic/infertility disorder) in females. Emerging clinical research has recently postulated the existence of a male PCOS phenotype, as it has been observed that the male relatives of women with PCOS manifest an array of similar reproductive and metabolic abnormalities. Therefore, this project aimed to elucidate the reproductive and metabolic phenotype of prenatally androgenised mice (PNAM).

Pregnant mouse dams were subcutaneously injected with either 250µg of dihydrotestosterone (DHT) (eliciting PNAM offspring) or a control oil vehicle solution (200µL) (vehicle control offspring) on gestational days 16, 17 and 18. Pubertal onset and fertility was analysed by determining the age of the first successful mating (resulting in conception) and daily anogenital distance (the distance between the glans penis and anus) measurements from postnatal day 35 onwards. Body weight measurements were recorded every five days throughout puberty and then every 10 days throughout early adulthood. Testes and seminal vesicles weight was additionally measured.

Overall, this project found no statistically significant differences between PNAM and vehicle controls in terms of body weight, age of first successful mating and seminal vesicles weight. However, the normalised anogenital distance measurement by body weight of PNAM (n=20) mice was significantly shorter (ie, female-like and suggestive decreased circulating androgens) at postnatal day 40 in comparison to vehicle controls (n=15) (P<0.01). Additionally, the normalised testes to body weight ratio was significantly lower in PNAM mice (Mann Whitney U test, P=0.0152).

Therefore, this study has illustrated males exposed to elevated prenatal androgens manifest small changes in external genitalia and gonadal weight, but are still fertile and exhibit no overt metabolic phenotype. It remains to be determined whether men exposed to elevated maternal androgens manifest any reproductive abnormalities.

Supported by a Division of Health Sciences Summer Research Scholarship.

Folia-specific vulnerability of the cerebellar climbing fibre synapse in a mouse model of human spinocerebellar ataxia type 1

EA Deeney, RM Empson

Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin.

Ataxia, or loss of movement control, is caused by disrupted cerebellar synaptic connections. The cerebellar cortex exhibits a foliated anatomical structure (undergoes repeated folding) that is largely conserved between mice and humans. All folia contain Purkinje neurons (PNs) that receive strong excitatory climbing fibre (CF) synaptic connections that modulate their output, but how this CF synaptic organisation differs in health and disease remains unknown. PNs can be sub-grouped based on molecular markers they express, such as excitatory amino acid transporter, EAAT4, which varies across folia. Furthermore, reduced EAAT4 expression and disrupted CF morphology both occur in the early stages of spinocerebellar ataxia type 1 (SCA1). Here, we test the hypothesis that CF disruption in SCA1 is greatest in cerebellar folia where EAAT4 is normally expressed.

Sagittal cerebellar sections (30µm) were collected from 15-week-old ataxic SCA1 and non-ataxic wild-type (WT) mice for fluorescence immunohistochemistry and confocal microscopy. Double-labelling with EAAT4 and calbindin ensured co-localisation at PNs, while calbindin and vesicular glutamate transporter 2 were used to assess PN and CF morphology, respectively.

In WT mice we observed higher EAAT4 expression in posterior versus anterior cerebellar folia (P<0.01, one-way ANOVA, n=4). CF morphology also varied across folia in WT mice (anterior versus posterior, P<0.001, two-way ANOVA, Tukey’s multiple comparisons), but CF morphology in SCA1 mice was only disrupted in posterior folia (interaction P<0.001, two-way ANOVA, n=6 WT, SCA1). However, SCA1 mice showed PN atrophy throughout the cerebellum, compared to WT mice (P<0.001, two-way ANOVA, n=6 WT, SCA1).

Results indicate that CF synapses located in the posterior cerebellum are more vulnerable to loss of EAAT4 expression in SCA1 mice. Our findings suggest the need for folia-specific therapeutic targeting for early treatment of SCA1.

Supported by a Division of Health Sciences Summer Research Scholarship.

Do online resources foster self-management support in people with persistent pain?

Devan H,1 Perry MA,1 Farmery D,2 Cotes L,2 van Hattem A,3 Thurlow G,3 Shepherd S,3 Muchemwa C,3 Grainger R4

1Centre for Health, Activity, and Rehabilitation Research (CHARR), School of Physiotherapy, University of Otago, Dunedin; 2Otago Medical School, University of Otago, Dunedin; 3School of Physiotherapy, University of Otago, Dunedin; 4Department of Medicine, University of Otago, Wellington.

Persistent non-cancer pain affects one in five New Zealanders. Online resources such as smartphone applications (apps) and websites are a potential solution for fostering self-management skills such as self-reflection and active goal setting in people with persistent pain. The aim of our review was to evaluate the contents of online resources (apps and websites) for people with persistent pain facilitating self-management support.

A systematic search was performed in the New Zealand App Store and Google Play Store and major search engines Google, Bing and Yahoo. Online resources were included if they were designed for people with persistent pain, provided information on pain management strategies and available in English. The contents were evaluated using an a priori 14-item self-management support checklist. The 23-item Mobile Apps Rating Scale (MARS) and HONcode certification was used to appraise app and website quality respectively.

Eighteen apps and 27 websites met inclusion criteria. Overall, the included apps and websites met a median of 3 (range 1–8) and 5 (range 1–8) of the 14-item self-management checklist. For both apps and websites, self-monitoring of symptoms and self-tailoring of strategies were frequently featured functions, while few online resources had features facilitating social support and communicating with clinicians. None provided information tailored to cultural needs of the user. The app quality mean scores using MARS ranged from 2.7 to 4.5 (out of 5.0). HONcode certification was present in six of the 27 websites. Only two apps and two websites underwent scientific evaluation supporting their clinical efficacy.

Although pain management apps and websites provide information to develop self-efficacy, none provided culturally appropriate information. While few apps and websites were validated to show improved health outcomes, none were tested in people with persistent pain. Both users and clinicians have to be aware of such limitations and make informed choices in using and recommending online resources as a self-management tool.

Supported by grants from Arthritis New Zealand and School of Physiotherapy, University of Otago.

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