Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

Investigating the effects of preinduction on human adipose-derived precursor cells in an athymic rat model

The osteogenic potential of human adipose-derived precursor cells seeded on medical-grade polycaprolactone-tricalcium phosphate scaffolds was investigated in this in vivo study. Three study groups were investigated: (1) induced—stimulated with osteogenic factors only after seeding into scaffold; (2) preinduced—induced for 2 weeks before seeding into scaffolds; and (3) uninduced—cells without any introduced induction. For all groups, scaffolds were implanted subcutaneously into the dorsum of athymic rats. The scaffold/cell constructs were harvested at the end of 6 or 12 weeks and analyzed for osteogenesis. Gross morphological examination using scanning electron microscopy indicated good integration of host tissue with scaffold/cell constructs and extensive tissue infiltration into the scaffold interior. Alizarin Red histology and immunostaining showed a heightened level of mineralization and an increase in osteonectin, osteopontin, and collagen type I protein expression in both the induced and preinduced groups compared with the uninduced groups. However, no significant differences were observed in these indicators when compared between the induced and preinduced groups.

Migration of breast cancer cells : understanding the roles of volume exclusion and cell-to-cell adhesion

We study MCF-7 breast cancer cell movement in a transwell apparatus. Various experimental conditions lead to a variety of monotone and nonmonotone responses which are difficult to interpret. We anticipate that the experimental results could be caused by cell-to-cell adhesion or volume exclusion. Without any modeling, it is impossible to understand the relative roles played by these two mechanisms. A lattice-based exclusion process random-walk model incorporating agent-to-agent adhesion is applied to the experimental system. Our combined experimental and modeling approach shows that a low value of cell-to-cell adhesion strength provides the best explanation of the experimental data suggesting that volume exclusion plays a more important role than cell-to-cell adhesion. This combined experimental and modeling study gives insight into the cell-level details and design of transwell assays.

Selective accumulation of virus-specific CD8+ T cells within the peripheral blood stem cell compartment

The absence of cellular immunity is central to the pathogenesis of herpesvirus-mediated diseases after allogeneic hemopoietic stem cell transplantation (HSCT). For both bone marrow (BM)– and granulocyte-colony stimulating factor–mobilized peripheral blood stem cells (PBSCs) HSCT, donor-derived Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide–specific CD8+ T cells clones undergo early expansion and persist long-term, with additional diversification arising from novel antigen-specific clones from donor-derived progenitors. Whether BM or PBSC is the superior source of antiviral CD8+ T cells is unclear. Given that PBSC has largely replaced BM as a source of stem cells for HSCT, it is unlikely that herpesvirus effector T-cell reconstitution will ever be compared prospectively. PBSC grafts contain 10 to 30 times more T cells than BM and a randomized study found proven viral infections were more frequent in BM than PBSC recipients, suggesting viral-specific T-cell immunity is enhanced in PBSC. Recently Moss showed in lung cancer patients that herpesvirus-specific BM-derived CD8+ T cells have unique homing properties relative to herpesvirus-specific CD8+ T cells present in unmobilized peripheral blood (PB). Immunodominant EBV-lytic peptide–specific CD8+ T cells were enriched in BM but were reduced for CMV peptide–specific CD8+ T cells relative to PB. EBV-latent peptide–specific CD8+ T cells were equivalent, which has relevance in the context of posttransplantation lymphoproliferative disorder for which impaired EBV-latent CD8+ T-cell immunity is a risk-factor. A comparison of herpesvirus-specific cellular immunity in PBSC versus PB has yet to be performed.

Ovine bone and marrow derived progenitor cells : isolation, characterization, and osteogenic potential

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) for their potential clinical use in bone engineering. Prior to clinical application, MPC-based treatment concepts need to be evaluated in preclinical, immunocompetent, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, ovine MPC and their osteogenic potential remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than osteoblasts (OB) derived from tibial compact bone as assessed using standard 2D culture. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14-/CD31-/CD45- /CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed >90%. Cells displayed a spindle-like morphology and formed an interconnected network. Implanted subcutaneously into NOD/SCID mice on type I collagen coated polycaprolactone-tricalciumphosphate (mPCL-TCP) scaffolds, MPC presented a higher developmental potential than osteoblasts. In summary, this study provides a detailed in vitro characterisation of ovine MPC from a bone engineering perspective and suggests that MPC provide promising means for future bone disease related treatment applications.

Contracting bubbles in Hele-Shaw cells with a power-law fluid

The problem of bubble contraction in a Hele-Shaw cell is studied for the case in which the surrounding fluid is of power-law type. A small perturbation of the radially symmetric problem is first considered, focussing on the behaviour just before the bubble vanishes, it being found that for shear-thinning fluids the radially symmetric solution is stable, while for shear-thickening fluids the aspect ratio of the bubble boundary increases. The borderline (Newtonian) case considered previously is neutrally stable, the bubble boundary becoming elliptic in shape with the eccentricity of the ellipse depending on the initial data. Further light is shed on the bubble contraction problem by considering a long thin Hele-Shaw cell: for early times the leading-order behaviour is one-dimensional in this limit; however, as the bubble contracts its evolution is ultimately determined by the solution of a Wiener-Hopf problem, the transition between the long-thin limit and the extinction limit in which the bubble vanishes being described by what is in effect a similarity solution of the second kind. This same solution describes the generic (slit-like) extinction behaviour for shear-thickening fluids, the interface profiles that generalise the ellipses that characterise the Newtonian case being constructed by the Wiener-Hopf calculation.

A chimeric vitronectin : IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells

This paper was retracted by the Journal of Stem Cells and Development on February 15, 2013.

Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds

Human mesenchymal stem cells (hMSCs) possess great therapeutic potential for the treatment of bone disease and fracture non-union. Too often however, in vitro evidence alone of the interaction between hMSCs and the biomaterial of choice is used as justification for continued development of the material into the clinic. Clearly for hMSC-based regenerative medicine to be successful for the treatment of orthopaedic trauma, it is crucial to transplant hMSCs with a suitable carrier that facilitates their survival, optimal proliferation and osteogenic differentiation in vitro and in vivo. This motivated us to evaluate the use of polycaprolactone-20% tricalcium phosphate (PCL-TCP) scaffolds produced by fused deposition modeling for the delivery of hMSCs. When hMSCs were cultured on the PCL-TCP scaffolds and imaged by a combination of phase contrast, scanning electron and confocal laser microscopy, we observed five distinct stages of colonization over a 21-day period that were characterized by cell attachment, spreading, cellular bridging, the formation of a dense cellular mass and the accumulation of a mineralized extracellular matrix when induced with osteogenic stimulants. Having established that PCL-TCP scaffolds are able to support hMSC proliferation and osteogenic differentiation, we next tested the in vivo efficacy of hMSC-loaded PCL-TCP scaffolds in nude rat critical-sized femoral defects. We found that fluorescently labeled hMSCs survived in the defect site for up to 3 weeks post-transplantation. However, only 50% of the femoral defects treated with hMSCs responded favorably as determined by new bone volume. As such, we show that verification of hMSC viability and differentiation in vitro is not sufficient to predict the efficacy of transplanted stem cells to consistently promote bone formation in orthotopic defects in vivo.

The application of a profluorescent nitroxide probe to detect reactive oxygen species derived from combustion-generated particulate matter

Particulate pollution has been widely recognised as an important risk factor to human health. In addition to increases in respiratory and cardiovascular morbidity associated with exposure to particulate matter (PM), WHO estimates that urban PM causes 0.8 million premature deaths globally and that 1.5 million people die prematurely from exposure to indoor smoke generated from the combustion of solid fuels. Despite the availability of a huge body of research, the underlying toxicological mechanisms by which particles induce adverse health effects are not yet entirely understood. Oxidative stress caused by generation of free radicals and related reactive oxygen species (ROS) at the sites of deposition has been proposed as a mechanism for many of the adverse health outcomes associated with exposure to PM. In addition to particle-induced generation of ROS in lung tissue cells, several recent studies have shown that particles may also contain ROS. As such, they present a direct cause of oxidative stress and related adverse health effects. Cellular responses to oxidative stress have been widely investigated using various cell exposure assays. However, for a rapid screening of the oxidative potential of PM, less time-consuming and less expensive, cell-free assays are needed. The main aim of this research project was to investigate the application of a novel profluorescent nitroxide probe, synthesised at QUT, as a rapid screening assay in assessing the oxidative potential of PM. Considering that this was the first time that a profluorescent nitroxide probe was applied in investigating the oxidative stress potential of PM, the proof of concept regarding the detection of PM–derived ROS by using such probes needed to be demonstrated and a sampling methodology needed to be developed. Sampling through an impinger containing profluorescent nitroxide solution was chosen as a means of particle collection as it allowed particles to react with the profluorescent nitroxide probe during sampling, avoiding in that way any possible chemical changes resulting from delays between the sampling and the analysis of the PM. Among several profluorescent nitroxide probes available at QUT, bis(phenylethynyl)anthracene-nitroxide (BPEAnit) was found to be the most suitable probe, mainly due to relatively long excitation and emission wavelengths (λex= 430 nm; λem= 485 and 513 nm). These wavelengths are long enough to avoid overlap with the background fluorescence coming from light absorbing compounds which may be present in PM (e.g. polycyclic aromatic hydrocarbons and their derivatives). Given that combustion, in general, is one of the major sources of ambient PM, this project aimed at getting an insight into the oxidative stress potential of combustion-generated PM, namely cigarette smoke, diesel exhaust and wood smoke PM. During the course of this research project, it was demonstrated that the BPEAnit probe based assay is sufficiently sensitive and robust enough to be applied as a rapid screening test for PM-derived ROS detection. Considering that for all three aerosol sources (i.e. cigarette smoke, diesel exhaust and wood smoke) the same assay was applied, the results presented in this thesis allow direct comparison of the oxidative potential measured for all three sources of PM. In summary, it was found that there was a substantial difference between the amounts of ROS per unit of PM mass (ROS concentration) for particles emitted by different combustion sources. For example, particles from cigarette smoke were found to have up to 80 times less ROS per unit of mass than particles produced during logwood combustion. For both diesel and wood combustion it has been demonstrated that the type of fuel significantly affects the oxidative potential of the particles emitted. Similarly, the operating conditions of the combustion source were also found to affect the oxidative potential of particulate emissions. Moreover, this project has demonstrated a strong link between semivolatile (i.e. organic) species and ROS and therefore, clearly highlights the importance of semivolatile species in particle-induced toxicity.

Solving navigational uncertainty using grid cells on robots

To successfully navigate their habitats, many mammals use a combination of two mechanisms, path integration and calibration using landmarks, which together enable them to estimate their location and orientation, or pose. In large natural environments, both these mechanisms are characterized by uncertainty: the path integration process is subject to the accumulation of error, while landmark calibration is limited by perceptual ambiguity. It remains unclear how animals form coherent spatial representations in the presence of such uncertainty. Navigation research using robots has determined that uncertainty can be effectively addressed by maintaining multiple probabilistic estimates of a robot's pose. Here we show how conjunctive grid cells in dorsocaudal medial entorhinal cortex (dMEC) may maintain multiple estimates of pose using a brain-based robot navigation system known as RatSLAM. Based both on rodent spatially-responsive cells and functional engineering principles, the cells at the core of the RatSLAM computational model have similar characteristics to rodent grid cells, which we demonstrate by replicating the seminal Moser experiments. We apply the RatSLAM model to a new experimental paradigm designed to examine the responses of a robot or animal in the presence of perceptual ambiguity. Our computational approach enables us to observe short-term population coding of multiple location hypotheses, a phenomenon which would not be easily observable in rodent recordings. We present behavioral and neural evidence demonstrating that the conjunctive grid cells maintain and propagate multiple estimates of pose, enabling the correct pose estimate to be resolved over time even without uniquely identifying cues. While recent research has focused on the grid-like firing characteristics, accuracy and representational capacity of grid cells, our results identify a possible critical and unique role for conjunctive grid cells in filtering sensory uncertainty. We anticipate our study to be a starting point for animal experiments that test navigation in perceptually ambiguous environments.

Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay

Reviewing the available literature, one could conclude that marrow-derived mesenchymal stem cells (BMSCs) are the ‘gold standard’ source for bone tissue engineering applications, due to their multilineage differentiation potential and easy accessibility. However, comprehensive studies comparing their osteogenic potential with bone-derived osteoblasts (OBs) to justify the preferred application of BMSCs based on performance are few. To address these shortfalls, in the present study, ovine BMSCs and OBs seeded onto scaffolds were characterized in vitro and transplanted subcutaneously into NOD/SCID mice in combination with and without recombinant human bone morphogenetic protein 7 (rhBMP-7). It was hypothesized that cell origin, ossification type and degree of vascularization and ossification depends on the nature and commitment of transplanted cells and stimulating growth factors, such as rhBMP-7. After retrieval, specimens were analysed by biomechanical testing, µCT analysis, scanning electron microscopy/energy-dispersive X-ray spectroscopy and histo- and immunohistochemistry for osteocalcin, type II collagen and BrdU. The results showed a high degree of cell survival and proliferation ectopically, resulting in active contribution to endochondral osteogenesis. When compared to BMSCs, OBs showed a higher degree of bone deposition while OB-derived bone was of higher maturation. Stimulation with rhBMP-7 increased the rate of bone synthesis for both BMSCs and OBs, additionally promoting neovascularization and osteoclast activity. These results suggest that the origin and commitment of transplanted cells highly influence the type and degree of ossification, that rhBMP-7 represents a powerful adjuvant for bone tissue-engineering applications, and that mature bone is an adequate alternative cell source for bone tissue-engineering applications.

Apoptosis is induced in Chlamydia trachomatis infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.

Chlamydial infection of immune cells : altered function and implications for disease

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects the genital and ocular mucosa of humans, causing infections that can lead to pelvic inflammatory disease, infertility, and blinding trachoma. C. pneumoniae is a respiratory pathogen that is the cause of 12–15% of community-acquired pneumonia. Both chlamydial species were believed to be restricted to the epithelia of the genital, ocular, and respiratory mucosa; however, increasing evidence suggests that both these pathogens can be isolated from peripheral blood of both healthy individuals and patients with inflammatory conditions such as coronary artery disease and asthma. Chlamydia can also be isolated from brain tissues of patients with degenerative neurological disorders such as Alzheimer’s disease and multiple sclerosis, and also from certain lymphomas. An increasing number of in vitro studies suggest that some chlamydial species can infect immune cells, at least at low levels. These infections may alter immune cell function in a way that promotes chlamydial persistence in the host and contributes to the progression of several chronic inflammatory diseases. In this paper, we review the evidence for the growth of Chlamydia in immune cells, particularly monocytes/macrophages and dendritic cells, and describe how infection may affect the function of these cells.

The impact of sulphur content of diesel fuel on ultrafine particle formation

Early this year the Australian Department of Environment and Heritage commissioned a desktop literature review with a focus on ultrafine particles including analysis of health impacts of the particles as well as the impact of sulphur content of diesel fuel on ultrafine particle emission. This paper summarizes the findings of the report on the link between the sulphur content of diesel fuels and the number of ultrafine particles in diesel emissions. The literature search on this topic resulted in over 150 publications. The majority of these publications, although investigating different aspects of the influence of fuel sulphur level on diesel vehicle emissions, were not directly concerned with ultrafine particle emissions. A specific focus of the paper is on: ----- ----- summary of state of knowledge established by the review, and ----- ----- summary of recommendations on the research priorities for Australia to address the information gaps for this issue, and on the appropriate management responses.