In vitro physical stimulation of tissue-engineered and native cartilage

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

Evaluation of the Suchey-Brooks Method of age estimation in an Australian subpopulation using computed tomography of the pubic symphyseal surface

Despite the prominent use of the Suchey-Brooks (S-B) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential inter-population error rates between geographical locations. This study assessed the accuracy of the S-B method to a contemporary adult population in Queensland, Australia and provides robust age parameters calibrated for our population. Three-dimensional surface reconstructions were generated from computed tomography scans of the pubic symphysis of male and female Caucasian individuals aged 15–70 years (n = 195) in Amira® and Rapidform®. Error was analyzed on the basis of bias, inaccuracy and percentage correct classification for left and right symphyseal surfaces. Application of transition analysis and Chi-square statistics demonstrated 63.9% and 69.7% correct age classification associated with the left symphyseal surface of Australian males and females, respectively, using the S-B method. Using Bayesian statistics, probability density distributions for each S-B phase were calculated, providing refined age parameters for our population. Mean inaccuracies of 6.77 (±2.76) and 8.28 (±4.41) years were reported for the left surfaces of males and females, respectively; with positive biases for younger individuals (<55 years) and negative biases in older individuals. Significant sexual dimorphism in the application of the S-B method was observed; and asymmetry in phase classification of the pubic symphysis was a frequent phenomenon. These results recommend that the S-B method should be applied with caution in medico-legal death investigations of Queensland skeletal remains and warrant further investigation of reliable age estimation techniques.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Within-subject intra- and inter-method consistency of two experimental risk attitude elicitation methods

We compare the consistency of choices in two methods to used elicit risk preferences on an aggregate as well as on an individual level. We asked subjects to choose twice from a list of nine decision between two lotteries, as introduced by Holt and Laury (2002, 2005) alternating with nine decisions using the budget approach introduced by Andreoni and Harbaugh (2009). We find that while on an aggregate(subject pool) level the results are (roughly) consistent, on an individual(within-subject) level,behavior is far from consistent. Within each method as well as across methods we observe low correlations. This again questions the reliability of experimental risk elicitation measures and the ability to use results from such methods to control for the risk aversion of subjects when explaining e�ects in other experimental games.

An analytical method for assessing stage-specific drug activity in Plasmodium vivax malaria : implications for ex vivo drug susceptibility testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC50) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC50 for CQ. Our findings indicate that EC50 values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.

Suspected child abuse and neglect reporting behaviour

Executuve Summary Background and Aims Child abuse and neglect is a tragedy within our community, with over 10,000 substantiated reports of abuse and neglect in Queensland in the past year. The considerable consequences of child abuse and neglect are far-reaching, substantial and can be fatal. The reporting of suspicions of child abuse or neglect is often the first step in preventing further abuse or neglect. In the State of Queensland, medical practitioners are mandated by law to report their suspicions of child abuse and neglect. However, despite this mandate many still do not report their suspicions. A 1998 study indicated that 43% of medical practitioners had, at some time, made a conscious decision to not report suspected abuse or neglect (Van Haeringen, Dadds & Armstrong, 1998). The aim of this study was to gain a better understanding of beliefs about reporting suspected child abuse and neglect and the barriers to reporting suspected abuse and neglect by medical practitioners and parents and students. The findings have the potential to inform the training and education of members of the community who have a shared responsibility to protect the wellbeing of its most vulnerable members. Method In one of the largest studies of reporting behaviour in relation to suspected child abuse and neglect in Australia, we examined and compared medical practitioners’ responses with members of the community, namely parents and students. We surveyed 91 medical practitioners and 214 members of the community (102 parents and 112 students) regarding their beliefs and reporting behaviour related to suspected child abuse and neglect. We also examined reasons for not reporting suspected abuse or neglect, as well as awareness of responsibilities and the appropriate reporting procedures. To obtain such information, participants anonymously completed a comprehensive questionnaire using items from previous studies of reporting attitudes and behaviour. Executive Summary Abused Child Trust Report August 2003 5 Findings Key findings include: • The majority of medical practitioners (97%) were aware of their duty to report suspected abuse and neglect and believed they had a professional and ethical duty to do so. • A majority of parents (82%) and students (68%) also believed that they had a professional and ethical duty to report suspected abuse and neglect. • In accord with their statutory duty to report suspected abuse and neglect, 69% of medical practitioners had made a report at some point. • Sixteen percent of parents and 9% of students surveyed indicated that they had reported their suspicions of neglect and abuse. • The most endorsed belief associated with not reporting suspected child abuse and neglect was that, ‘unpleasant events would follow reporting’. • Over a quarter of medical practitioners (26%) admitted to making a decision not to report their suspicions of child abuse or neglect on at least one occasion. • Compared with previous research, there has been a decline in the number of medical practitioners who decided not to report suspected abuse or neglect from 43% (Van Haeringen et al., 1998) to 26% in the current study. • Fourteen percent of parents and 15% of students surveyed had also chosen not to report a case of suspected abuse or neglect. • Attitudes that most strongly influenced the decision to report or not report suspected abuse or neglect differed between groups (medical practitioners, parents, or students). A belief that, ‘the abuse was a single incident’ was the best predictor of non-reporting by medical practitioners, while having ‘no time to follow-up the report’ or failing to be ‘convinced of evidence of abuse’ best predicted failure to report abuse by students. A range of beliefs predicted non-reporting by parents, including the beliefs that reporting suspected abuse was ‘not their responsibility’ and ‘knowing the child had retracted their statement’. Conclusions Of major concern is that approximately 25% of medical practitioners with a mandated responsibility to report, as well as some members of the general public, revealed that they have suspected child neglect or abuse but have made the decision not to report their suspicions. Parents and students perceived the general community as having responsibility for reporting suspicions of abuse or neglect. Despite this perception, they felt that lodging a report may be overly demanding in terms of time and they had the confidence in their ability to identify child abuse and neglect. An explanation for medical practitioners deciding not to report may be based upon their optimistic belief that suspected abuse or neglect was a single incident. Our findings may best be understood from the ‘inflation of optimism’ hypothesis put forward by the Nobel Laureate, Daniel Kahneman. He suggests that in spite of rational evidence, human beings tend to make judgements based on an optimistic view rather than engaging in a rational decision-making process. In this case, despite past behaviour of abuse or neglect being the best predictor of future behaviour, medical practitioners have taken an optimistic view, choosing to believe that their suspicion of child abuse or neglect represents a single incident. The clear implication of findings in the current research is the need for the members of the general community and medical practitioners to be better appraised of the consequences of their decision-making in relation to suspicionsof child abuse and neglect. Finally findings from parents and students relating to their reporting behaviour suggest that members of the larger community represent an untapped resourcewho might, with appropriate awareness, play a more significant role in theidentification and reporting of suspected child abuse and neglect.

Contribution to single-point closure Reynolds-stress modelling of inhomogeneous flow

This paper is concerned with recent advances in the development of near wall-normal-free Reynolds-stress models, whose single point closure formulation, based on the inhomogeneity direction concept, is completely independent of the distance from the wall, and of the normal to the wall direction. In the present approach the direction of the inhomogeneity unit vector is decoupled from the coefficient functions of the inhomogeneous terms. A study of the relative influence of the particular closures used for the rapid redistribution terms and for the turbulent diffusion is undertaken, through comparison with measurements, and with a baseline Reynolds-stress model (RSM) using geometric wall normals. It is shown that wall-normal-free rsms can be reformulated as a projection on a tensorial basis that includes the inhomogeneity direction unit vector, suggesting that the theory of the redistribution tensor closure should be revised by taking into account inhomogeneity effects in the tensorial integrity basis used for its representation.

Numerical simulation of red blood cells' deformation using SPH method

A numerical simulation method for the Red Blood Cells’ (RBC) deformation is presented in this study. The two-dimensional RBC membrane is modeled by the spring network, where the elastic stretch/compression energy and the bending energy are considered with the constraint of constant RBC surface area. Smoothed Particle Hydrodynamics (SPH) method is used to solve the Navier-Stokes equation coupled with the Plasma-RBC membrane and Cytoplasm- RBC membrane interaction. To verify the method, the motion of a single RBC is simulated in Poiseuille flow and compared with the results reported earlier. Typical motion and deformation mechanism of the RBC is observed.