Orthopaedics and Trauma Queensland Annual Report 2011

Orthopaedics and Trauma Queensland, a Centre for Research and Education in Musculoskeletal Disorders, is an internationally recognised research group that is developing into an international leader in research and education. It provides a stimulus for research, education and clinical application within the international orthopaedic and trauma communities. Orthopaedics and Trauma Queensland develops and promotes the innovative use of engineering and technology, in collaboration with surgeons, to provide new techniques, materials, procedures and medical devices. Its integration with clinical practice and strong links with hospitals ensure that the research will be translated into practical outcomes for patients. The group undertakes clinical practice in orthopaedics and trauma and applies core engineering skills to challenges in medicine. The research is built on a strong foundation of knowledge in biomedical engineering, and incorporates expertise in cell biology, mathematical modelling, human anatomy and physiology and clinical medicine in orthopaedics and trauma. New knowledge is being developed and applied to the full range of orthopaedic diseases and injuries, such as knee and hip replacements, fractures and spinal deformities.

Quantifying the roles of cell motility and cell proliferation in a circular barrier assay

Moving fronts of cells are essential features of embryonic development, wound repair and cancer metastasis. This paper describes a set of experiments to investigate the roles of random motility and proliferation in driving the spread of an initially confined cell population. The experiments include an analysis of cell spreading when proliferation was inhibited. Our data have been analysed using two mathematical models: a lattice-based discrete model and a related continuum partial differential equation model. We obtain independent estimates of the random motility parameter, D, and the intrinsic proliferation rate, λ, and we confirm that these estimates lead to accurate modelling predictions of the position of the leading edge of the moving front as well as the evolution of the cell density profiles. Previous work suggests that systems with a high λ/D ratio will be characterized by steep fronts, whereas systems with a low λ/D ratio will lead to shallow diffuse fronts and this is confirmed in the present study. Our results provide evidence that continuum models, based on the Fisher–Kolmogorov equation, are a reliable platform upon which we can interpret and predict such experimental observations.

Morphology-based model for predicting osteocyte-like cell line (MLO-Y4) growth process

This work has led to the development of empirical mathematical models to quantitatively predicate the changes of morphology in osteocyte-like cell lines (MLO-Y4) in culture. MLO-Y4 cells were cultured at low density and the changes in morphology recorded over 11 hours. Cell area and three dimensional shape features including aspect ratio, circularity and solidity were then determined using widely accepted image analysis software (ImageJTM). Based on the data obtained from the imaging analysis, mathematical models were developed using the non-linear regression method. The developed mathematical models accurately predict the morphology of MLO-Y4 cells for different culture times and can, therefore, be used as a reference model for analyzing MLO-Y4 cell morphology changes within various biological/mechanical studies, as necessary.

An in-vitro 3-D cell culture model for studying pathomechanisms in AMD

Purpose To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin used in conjunction with cultured human cells can be used to mimic the structural relationships between the RPE and choriocapillaris in health and disease. Methods Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a synthetic Bruch’s membrane (3 microns thick) constructed from B mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. The effects of tropoelastin on attachment of ARPE-19 cells was also examined. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the synthetic RPE was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing within a 24 G needle. The architecture of the synthetic tissue before and after wounding was examined by confocal microscopy after staining for ZO-1 and F-actin. Results The RPE layer of the 3D model developed a cobblestoned morphology (validated by staining for ZO-1 and F-actin), displayed barrier function (validated by measurement of TER) and demonstrated cytoplasmic uptake of vitronectin-coated microspheres. Attachment of ARPE-19 cells to fibroin was unaffected by tropoelastin. Microvascular endothelial cells attached well to the gelatin-coated surface of the fibroin membrane and remained physically separated from the overlaying RPE layer. The fibroin membranes were amenable to puncturing without collapse thus providing the opportunity to study transmembrane migration of the endothelial cells. Conclusions Synthetic Bruch’s membranes constructed from silk fibroin, vitronectin and gelatin, support the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model of choroidal neovascularization.

Temporal summation of intrinsically photosensitive retinal ganglion cell (ipRGC) contributions to the human pupil.

Purpose: IpRGCs mediate non-image forming functions including photoentrainment and the pupil light reflex (PLR). Temporal summation increases visual sensitivity and decreases temporal resolution for image forming vision, but the summation properties of nonimage forming vision are unknown. We investigated the temporal summation of inner (ipRGC) and outer (rod/cone) retinal inputs to the PLR. Method: The consensual PLR of the left eye was measured in six participants with normal vision using a Maxwellian view infrared pupillometer. Temporal summation was investigated using a double-pulse protocol (100 ms stimulus pairs; 0–1024 ms inter-stimulus interval, ISI) presented to the dilated fellow right eye (Tropicamide 1%). Stimulus lights (blue λmax = 460 nm; red λmax = 638 nm) biased activity to inneror outer retinal inputs to non-image forming vision. Temporal summation was measured suprathreshold (15.2 log photons.cm−2.s−1 at the cornea) and subthreshold (11.4 log photons.cm−2.s−1 at the cornea). Results: RM-ANOVAs showed the suprathreshold and subthreshold 6 second post illumination pupil response (PIPR: expressed as percentage baseline diameter) did not significantly vary for red or blue stimuli (p > .05). The PIPR for a subthreshold red 16 ms double-pulse control condition did not significantly differ with ISI (p > .05). The maximum constriction amplitude for red and blue 100 ms double- pulse stimuli did not significantly vary with ISI (p > .05). Conclusion: The non-significant changes in suprathreshold PIPR and subthreshold maximum pupil constriction indicate that inner retinal ipRGC inputs and outer retinal photoreceptor inputs to the PLR do not show temporal summation. The results suggest a fundamental difference between the temporal summation characteristics of image forming and non-image forming vision.

Cell death control : the interplay of apoptosis and autophagy in the pathogenicity of sclerotinia sclerotiorum

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.

Travelling waves for a velocity–jump model of cell migration and proliferation

Cell invasion, characterised by moving fronts of cells, is an essential aspect of development, repair and disease. Typically, mathematical models of cell invasion are based on the Fisher–Kolmogorov equation. These traditional parabolic models can not be used to represent experimental measurements of individual cell velocities within the invading population since they imply that information propagates with infinite speed. To overcome this limitation we study combined cell motility and proliferation based on a velocity–jump process where information propagates with finite speed. The model treats the total population of cells as two interacting subpopulations: a subpopulation of left–moving cells, $L(x,t)$, and a subpopulation of right–moving cells, $R(x,t)$. This leads to a system of hyperbolic partial differential equations that includes a turning rate, $\Lambda \ge 0$, describing the rate at which individuals in the population change direction of movement. We present exact travelling wave solutions of the system of partial differential equations for the special case where $\Lambda = 0$ and in the limit that $\Lambda \to \infty$. For intermediate turning rates, $0 < \Lambda < \infty$, we analyse the travelling waves using the phase plane and we demonstrate a transition from smooth monotone travelling waves to smooth nonmonotone travelling waves as $\Lambda$ decreases through a critical value $\Lambda_{crit}$. We conclude by providing a qualitative comparison between the travelling wave solutions of our model and experimental observations of cell invasion. This comparison indicates that the small $\Lambda$ limit produces results that are consistent with experimental observations.

Reduction in resting plasma granulysin as a marker of increased training load

Granulysin is a cytolytic granule protein released by natural killer cells and activated cytotoxic T lymphocytes. The influence of exercise training on circulating granulysin concentration is unknown, as is the relationship between granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. We examined changes in plasma granulysin concentration, natural killer cell number and cytotoxicity following acute exercise and different training loads. Fifteen highly trained male cyclists completed a baseline 40-km cycle time trial (TT401) followed by five weeks of normal training and a repeat time trial (TT402). The cyclists then completed four days of high intensity training followed by another time trial (TT403) on day five. Following one final week of normal training cyclists completed another time trial (TT404). Fasting venous blood was collected before and after each time trial to determine granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. Granulysin concentration increased significantly after each time trial (P<0.001). Pre-exercise granulysin concentration for TT403 was significantly lower than pre-exercise concentration for TT401 (-20.3 +/- 7.5%, P<0.026), TT402 (-16.7 +/- 4.3%, P<0.003) and 7T404 (-21 +/- 4.2%, P<0.001). Circulating natural killer cell numbers also increased significantly post-exercise for each time trial (P<0.001), however there was no significant difference across TT40 (P>0.05). Exercise did not significantly alter natural killer cell cytotoxicity on a per cell basis, and there were no significant differences between the four time trials. In conclusion, plasma granulysin concentration increases following moderate duration, strenuous exercise and is decreased in response to a short-term period of intensified training.

Exercise-recruited NK cells display exercise-associated eHSP-70

It is hypothesized that increased plasma or serum concentrations of extracellular heat shock proteins (eHSP) serve as a danger signal to the innate immune system. Cellular binding of eHSP leads to activation of NK cells and monocytes, as measured by their increased cytokine production, mitotic division and killing capacity. We examined whether eHSP binds to NK lymphocytes in vivo in athletes performing endurance exercise in the heat. Eighteen trained male runners ran at 70% VO2max at 35 degrees C and 40% relative humidity. Venous blood collected before, after and 1.5 h after exercise was analysed for leukocyte distribution, phenotype and eHSP70. NK cell-enriched samples were examined for co-localization of CD94 and eHSP70 expression. Plasma eHSP-70 concentration was measured by ELISA. Subjects ran for approximately 50 min, which elicited a reversible leukocytosis. NK cell count increased 83% (p < 0.01) immediately after exercise, then decreased to 66% of the resting level 1.5 h after exercise (p < 0.05). Plasma eHSP concentration increased 167% after exercise and remained elevated (by up to 71%) 1.5 h after exercise (p < 0.01). eHSP was expressed on both NK cells and monocytes at all times; the count of NK cells positive for eHSP doubled from 0.04 +/- 0.02 10(9)/L (mean +/- SD) to 0.08 +/- 0.06 10(9)/L after exercise. In summary, exercise in the heat increased free plasma eHSP concentration, and the eHSP co-localized with CD94 on NK cells. These data confirm the link between exercise and activation of the innate immune system.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Exercise-induced alterations in neutrophil degranulation and respiratory burst activity : possible mechanisms of action

Neutrophils constitute 50-60% of all circulating leukocytes; they present the first line of microbicidal defense and are involved in inflammatory responses. To examine immunocompetence in athletes, numerous studies have investigated the effects of exercise on the number of circulating neutrophils and their response to stimulation by chemotactic stimuli and activating factors. Exercise causes a biphasic increase in the number of neutrophils in the blood, arising from increases in catecholamine and cortisol concentrations. Moderate intensity exercise may enhance neutrophil respiratory burst activity, possibly through increases in the concentrations of growth hormone and the inflammatory cytokine IL-6. In contrast, intense or long duration exercise may suppress neutrophil degranulation and the production of reactive oxidants via elevated circulating concentrations of epinephrine (adrenaline) and cortisol. There is evidence of neutrophil degranulation and activation of the respiratory burst following exercise-induced muscle damage. In principle, improved responsiveness of neutrophils to stimulation following exercise of moderate intensity could mean that individuals participating in moderate exercise may have improved resistance to infection. Conversely, competitive athletes undertaking regular intense exercise may be at greater risk of contracting illness. However, there are limited data to support this concept. To elucidate the cellular mechanisms involved in the neutrophil responses to exercise, researchers have examined changes in the expression of cell membrane receptors, the production and release of reactive oxidants and more recently, calcium signaling. The investigation of possible modifications of other signal transduction events following exercise has not been possible because of current methodological limitations. At present, variation in exercise-induced alterations in neutrophil function appears to be due to differences in exercise protocols, training status, sampling points and laboratory assay techniques.

An accurate numerical scheme for the contraction of a bubble in a Hele-Shaw cell

We report on an accurate numerical scheme for the evolution of an inviscid bubble in radial Hele-Shaw flow, where the nonlinear boundary effects of surface tension and kinetic undercooling are included on the bubble-fluid interface. As well as demonstrating the onset of the Saffman-Taylor instability for growing bubbles, the numerical method is used to show the effect of the boundary conditions on the separation (pinch-off) of a contracting bubble into multiple bubbles, and the existence of multiple possible asymptotic bubble shapes in the extinction limit. The numerical scheme also allows for the accurate computation of bubbles which pinch off very close to the theoretical extinction time, raising the possibility of computing solutions for the evolution of bubbles with non-generic extinction behaviour.

Sensitivity of edge detection methods for quantifying cell migration assays

Quantitative imaging methods to analyze cell migration assays are not standardized. Here we present a suite of two–dimensional barrier assays describing the collective spreading of an initially–confined population of 3T3 fibroblast cells. To quantify the motility rate we apply two different automatic image detection methods to locate the position of the leading edge of the spreading population after 24, 48 and 72 hours. These results are compared with a manual edge detection method where we systematically vary the detection threshold. Our results indicate that the observed spreading rates are very sensitive to the choice of image analysis tools and we show that a standard measure of cell migration can vary by as much as 25% for the same experimental images depending on the details of the image analysis tools. Our results imply that it is very difficult, if not impossible, to meaningfully compare previously published measures of cell migration since previous results have been obtained using different image analysis techniques and the details of these techniques are not always reported. Using a mathematical model, we provide a physical interpretation of our edge detection results. The physical interpretation is important since edge detection algorithms alone do not specify any physical measure, or physical definition, of the leading edge of the spreading population. Our modeling indicates that variations in the image threshold parameter correspond to a consistent variation in the local cell density. This means that varying the threshold parameter is equivalent to varying the location of the leading edge in the range of approximately 1–5% of the maximum cell density.

SECIS-binding protein 2 promotes cell survival by protecting against oxidative stress

Reactive oxygen species (ROS) are a primary cause of cellular damage that leads to cell death. In cells, protection from ROS-induced damage and maintenance of the redox balance is mediated to a large extent by selenoproteins, a distinct family of proteins that contain selenium in form of selenocysteine (Sec) within their active site. Incorporation of Sec requires the Sec-insertion sequence element (SECIS) in the 3'-untranslated region of selenoproteins mRNAs and the SECIS-binding protein 2 (SBP2). Previous studies have shown that SBP2 is required for the Sec-incorporation mechanism; however, additional roles of SBP2 in the cell have remained undefined. We herein show that depletion of SBP2 by using antisense oligonucleotides (ASOs) causes oxidative stress and induction of caspase- and cytochrome c-dependent apoptosis. Cells depleted of SBP2 have increased levels of ROS, which lead to cellular stress manifested as 8-oxo-7,8-dihydroguanine (8-oxo-dG) DNA lesions, stress granules, and lipid peroxidation. Small-molecule antioxidants N-acetylcysteine, glutathione, and α-tocopherol only marginally reduced ROS and were unable to rescue cells fully from apoptosis, indicating that apoptosis might be directly mediated by selenoproteins. Our results demonstrate that SBP2 is required for protection against ROS-induced cellular damage and cell survival. Antioxid. Redox Signal. 12, 797–808.