Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia

Introduction Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. Methodology/Principal Findings Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. Conclusions/Significance Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

Mesenchymal stem cell therapies for bone and tendon conditions

Bone, tendon, and cartilage are highly specialized musculoskeletal connective tissues that are subject to injury and degeneration. These tissues have relatively poor healing capabilities, and coupled with their variable response to established medical treatments, produce significant morbidity. Mesenchymal stem cells (MSCs) are capable of regenerating skeletal tissues and therefore offer great promise in the treatment of connective tissue pathologies. Adult MSCs are multipotent cells that possess the properties of proliferation and differentiation into all connective tissues. Furthermore, they can be gene modified to secrete growth factors and utilized in connective tissue engineering. Potential MSC-based therapies for bone and tendon conditions are reviewed in this chapter.

Mesenchymal stromal cells and the repair of cartilage tissue

Articular cartilage has a limited intrinsic repair capacity, and thus defects are more likely to further degrade rather than undergo spontaneous self-repair. Whilst a number of surgical techniques have been developed to repair cartilage defects, their efficacy is generally poor and total joint replacement remains the gold standard, albeit last resort, treatment option. Cell-based therapies hold the greatest promise, as they appear uniquely capable of generating de novo cartilage tissue. Two approved therapies (ACI and MACI) are based on the premise that the transplantation of ex vivo expanded autologous chondrocyte populations, harvested from a non-load bearing region of the same joint, could be utilized to effectively regenerate cartilage tissue in the primary defect site. These therapeutic strategies are partially limited by our inability to harvest and expand adequate numbers of autologous chondrocytes that retain the appropriate phenotype. By contrast, the harvest and expansion of large numbers of mesenchymal stem/stromal cells (MSC) derived from tissues such as bone marrow and adipose is comparatively straightforward and has become routine in laboratories worldwide. Additionally, our understanding of the biochemical and biophysical signals required to drive the chondrogenic differentiation of MSC is rapidly increasing. It is conceivable that in the near future MSC expansion and differentiation technologies will offer a means to generate sufficient cell numbers, of an appropriate phenotype, for use in cartilage defect repair. In this chapter we review the relative potential of MSC and their likely contribution to cartilage regeneration.

Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: The next step towards "top-down" lipidomics

The complete structural elucidation of complex lipids, including glycerophospholipids, using only mass spectrometry represents a major challenge to contemporary analytical technologies. Here, we demonstrate that product ions arising from the collision-induced dissociation (CID) of the [M + Na] + adduct ions of phospholipids can be isolated and subjected to subsequent gas-phase ozonolysis-known as ozone-induced dissociation (OzID)-in a linear ion-trap mass spectrometer. The resulting CID/OzID experiment yields abundant product ions that are characteristic of the acyl substitution on the glycerol backbone (i.e., sn-position). This approach is shown to differentiate sn-positional isomers, such as the regioisomeric phosphatidylcholine pair of PC 16:0/18:1 and PC 18:1/16:0. Importantly, CID/OzID provides a sensitive diagnostic for the existence of an isomeric mixture in a given sample. This is of very high value for the analysis of tissue extracts since CID/OzID analyses can reveal changes in the relative abundance of isomeric constituents even within different tissues from the same animal. Finally, we demonstrate the ability to assign carbon-carbon double bond positions to individual acyl chains at specific backbone positions by adding subsequent CID and/or OzID steps to the workflow and that this can be achieved in a single step using a hybrid triple quadrupole-linear ion trap mass spectrometer. This unique approach represents the most complete and specific structural analysis of lipids by mass spectrometry demonstrated to date and is a significant step towards comprehensive top-down lipidomics. This journal is © The Royal Society of Chemistry 2014. Grant Number ARC/DP0986628, ARC/FT110100249, ARC/LP110200648

Toll-like receptor and pro-inflammatory cytokine expression during prolonged hyperinsulinaemia in horses : implications for laminitis

Equine laminitis, a disease of the lamellar structure of the horse’s hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Tolllike receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-�), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-� and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-�, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.

Ethnographic notes on visualization practices in tissue engineering

Visual information is central to several of the scientific disciplines. This paper studies how scientists working in a multidisciplinary field produce scientific evidence through building and manipulating scientific visualizations. Using ethnographic methods, we studied visualization practices of eight scientists working in the domain of tissue engineering research. Tissue engineering is an upcoming field of research that deals with replacing or regenerating human cells, tissues, or organs to restore or establish normal function. We spent 3 months in the field, where we recorded laboratory sessions of these scientists and used semi-structured interviews to get an insight into their visualization practices. From our results, we elicit two themes characterizing their visualization practices: multiplicity and physicality. In this article, we provide several examples of scientists’ visualization practices to describe these two themes and show that multimodality of such practices plays an important role in scientific visualization.

Synergism between Wnt3a and heparin enhances osteogenesis via a phosphoinositide 3-kinase/Akt/RUNX2 pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Expression and in vitro functions of the ghrelin axis in endometrial cancer

Ghrelin is a peptide hormone produced in the stomach and a range of other tissues, where it has endocrine, paracrine and autocrine roles in both normal and disease states. Ghrelin has been shown to be an important growth factor for a number of tumours, including prostate and breast cancers. In this study, we examined the expression of the ghrelin axis (ghrelin and its receptor, the growth hormone secretagogue receptor, GHSR) in endometrial cancer. Ghrelin is expressed in a range of endometrial cancer tissues, while its cognate receptor, GHSR1a, is expressed in a small subset of normal and cancer tissues. Low to moderately invasive endometrial cancer cell lines were examined by RT-PCR and immunoblotting, demonstrating that ghrelin axis mRNA and protein expression correlate with differentiation status of Ishikawa, HEC1B and KLE endometrial cancer cell lines. Moreover, treatment with ghrelin potently stimulated cell proliferation and inhibited cell death. Taken together, these data indicate that ghrelin promotes the progression of endometrial cancer cells in vitro, and may contribute to endometrial cancer pathogenesis and represent a novel treatment target.

A simple method for EPID-based in vivo dosimetry for radiotherapy treatments of head-and-neck cancers

This study used a homogeneous water-equivalent model of an electronic portal imaging device (EPID), contoured as a structure in a radiotherapy treatment plan, to produce reference dose images for comparison with in vivo EPID dosimetry images. Head and neck treatments were chosen as the focus of this study, due to the heterogeneous anatomies involved and the consequent difficulty of rapidly obtaining reliable reference dose images by other means. A phantom approximating the size and heterogeneity of a typical neck, with a maximum radiological thickness of 8.5 cm, was constructed for use in this study. This phantom was CT scanned and a simple treatment including five square test fields and one off-axis IMRT field was planned. In order to allow the treatment planning system to calculate dose in a model EPID positioned a distance downstream from the phantom to achieve a source-to-detector distance (SDD) of 150 cm, the CT images were padded with air and the phantom’s “body” contour was extended to encompass the EPID contour. Comparison of dose images obtained from treatment planning calculations and experimental irradiations showed good agreement, with more than 90% of points in all fields passing a gamma evaluation, at γ (3%, 3mm )Similar agreement was achieved when the phantom was over-written with air in the treatment plan and removed from the experimental beam, suggesting that water EPID model at 150 cm SDD is capable of providing accurate reference images for comparison with clinical IMRT treatment images, for patient anatomies with radiological thicknesses ranging from 0 up to approximately 9 cm. This methodology therefore has the potential to be used for in vivo dosimetry during treatments to tissues in the neck as well as the oral and nasal cavities, in the head-and-neck region.

Prospects for studying how high-intensity compression waves cause damage in human blast injuries

Since World War I, explosions have accounted for over 70% of all injuries in conflict. With the development of improved personnel protection of the torso, improved medical care and faster aeromedical evacuation, casualties are surviving with more severe injuries to the extremities. Understanding the processes involved in the transfer of blast-induced shock waves through biological tissues is essential for supporting efforts aimed at mitigating and treating blast injury. Given the inherent heterogeneities in the human body, we argue that studying these processes demands a highly integrated approach requiring expertise in shock physics, biomechanics and fundamental biological processes. This multidisciplinary systems approach enables one to develop the experimental framework for investigating the material properties of human tissues that are subjected to high compression waves in blast conditions and the fundamental cellular processes altered by this type of stimuli. Ultimately, we hope to use the information gained from these studies in translational research aimed at developing improved protection for those at risk and improved clinical outcomes for those who have been injured from a blast wave.

Silicate-based bioceramics for periodontal regeneration

Periodontal disease is characterized by the destruction of the tissues that attach the tooth to the alveolar bone. Various methods for regenerative periodontal therapy including the use of barrier membranes, bone replacement grafts, and growth factor delivery have been investigated; however, true regeneration of periodontal tissue is still a significant challenge to scientists and clinicians. The focus on periodontal tissue engineering has shifted from attempting to recreate tissue replacements/constructs to the development of biomaterials that incorporate and release regulatory signals to achieve in situ periodontal regeneration. The release of ions and molecular cues from biomaterials may help to unlock latent regenerative potential in the body by regulating cell proliferation and differentiation towards different lineages (e.g. osteoblasts and cementoblasts). Silicate-based bioactive materials, including bioactive silicate glasses and ceramics, have become the materials of choice for periodontal regeneration, due to their favourable osteoconductivity and bioactivity. This article will focus on the most recent advances in the in vitro and in vivo biological application of silicate-based ceramics, specifically as it relates to periodontal tissue engineering.

Effect of culture conditions and calcium phosphate coating on ectopic bone formation

This study investigated the effect of a calcium phosphate (CaP) coating onto a polycaprolactone melt electrospun scaffold and in vitro culture conditions on ectopic bone formation in a subcutaneous rat model. The CaP coating resulted in an increased alkaline phosphatase activity (ALP) in ovine osteoblasts regardless of the culture conditions and this was also translated into higher levels of mineralisation. A subcutaneous implantation was performed and increasing ectopic bone formation was observed over time for the CaPcoated samples previously cultured in osteogenic media whereas the corresponding non-coated samples displayed a lag phase before bone formation occurred from 4 to 8 weeks post-implantation. Histology and immunohistochemistry revealed bone fill through the scaffolds 8 weeks post-implantation for coated and non-coated specimens and that ALP, osteocalcin and collagen 1 were present at the ossification front and in the bone tissues. Vascularisation in the vicinity of the bone tissues was also observed indicating that the newly formed bone was not deprived of oxygen and nutrients.We found that in vitro osteogenic induction was essential for achieving bone formation and CaP coating accelerated the osteogenic process. We conclude that high cell density and preservation of the collagenous and mineralised extracellular matrix secreted in vitro are factors of importance for ectopic bone formation.

Bone regeneration based on tissue engineering conceptions – a 21st century perspective

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteo- conductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineer- ing and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

Rational design of artificial cellular niches for tissue engineering

Tissue Engineering is a promising emerging field that studies the intrinsic regenerative potential of the human body and uses it to restore functionality of damaged organs or tissues unable of self-healing due to illness or ageing. In order to achieve regeneration using Tissue Engineering strategies, it is first necessary to study the properties of the native tissue and determine the cause of tissue failure; second, to identify an optimum population of cells capable of restoring its functionality; and third, to design and manufacture a cellular microenvironment in which those specific cells are directed towards the desired cellular functions. The design of the artificial cellular niche has a tremendous importance, because cells will feel and respond to both its biochemical and biophysical properties very differently. In particular, the artificial niche will act as a physical scaffold for the cells, allowing their three-dimensional spatial organization; also, it will provide mechanical stability to the artificial construct; and finally, it will supply biochemical and mechanical cues to control cellular growth, migration, differentiation and synthesis of natural extracellular matrix. During the last decades, many scientists have made great contributions to the field of Tissue Engineering. Even though this research has frequently been accompanied by vast investments during extended periods of time, yet too often these efforts have not been enough to translate the advances into new clinical therapies. More and more scientists in this field are aware of the need of rational experimental designs before carrying out complex, expensive and time-consuming in vitro and in vivo trials. This review highlights the importance of computer modeling and novel biofabrication techniques as critical key players for a rational design of artificial cellular niches in Tissue Engineering.