Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.

CD73 and CD29 concurrently mediate the mechanically induced decrease of migratory capacity of mesenchymal stromal cells

The assumption that mesenchymal stromal cell (MSC)-based therapies are capable of augmenting physiological regeneration processes has fostered intensive basic and clinical research activities. However, to achieve sustained therapeutic success in vivo, not only the biological, but also the mechanical microenvironment of MSCs during these regeneration processes needs to be taken into account. This is especially important for e.g., bone fracture repair, since MSCs present at the fracture site undergo significant biomechanical stimulation. This study has therefore investigated cellular characteristics and the functional behaviour of MSCs in response to mechanical loading. Our results demonstrated a reduced expression of MSC surface markers CD73 (ecto-5’-nucleotidase) and CD29 (integrin β1) after loading. On the functional level, loading led to a reduced migration of MSCs. Both effects persisted for a week after the removal of the loading stimulus. Specifi c inhibition of CD73/CD29 demonstrated their substrate dependent involvement in MSC migration after loading. These results were supported by scanning electron microscopy images and phalloidin staining of actin fi laments displaying less cell spreading, lamellipodia formation and actin accumulations. Moreover, focal adhesion kinase and Src-family kinases were identified as candidate downstream targets of CD73/CD29 that might contribute to the mechanically induced decrease in MSC migration. These results suggest that MSC migration is controlled by CD73 CD29, which in turn are regulated by mechanical stimulation of cells. We therefore speculate that MSCs migrate into the fracture site, become mechanically entrapped, and thereby accumulate to fulfil their regenerative functions.

Glycosaminoglycan and growth factor mediated murine calvarial cell proliferation

Understanding the complex mechanisms underlying bone remodeling is crucial to the development of novel therapeutics. Glycosaminoglycans (GAGs) localised to the extracellular matrix (ECM) of bone are thought to play a key role in mediating aspects of bone development. The influence of isolated GAGs was studied by utilising in vitro murine calvarial monolayer and organ culture model systems. Addition of GAG preparations extracted from the cell surface of human osteoblasts at high concentrations (5 microg/ml) resulted in decreased proliferation of cells and decreased suture width and number of bone lining cells in calvarial sections. When we investigated potential interactions between the growth factors fibroblast growth factor-2 (FGF2), bone morphogenic protein-2 (BMP2) and transforming growth factor-beta1 (TGFbeta1) and the isolated cell surface GAGs, differences between the two model systems emerged. The cell culture system demonstrated a potentiating role for the isolated GAGs in the inhibition of FGF2 and TGFbeta1 actions. In contrast, the organ culture system demonstrated an enhanced stimulation of TFGbeta1 effects. These results emphasise the role of the ECM in mediating the interactions between GAGs and growth factors during bone development and suggest the GAG preparations contain potent inhibitory or stimulatory components able to mediate growth factor activity.

Purification and characterization of heparan sulfate from human primary osteoblasts

Heparan sulfate (HS) is a linear, highly variable, highly sulfated glycosaminoglycan sugar whose biological activity largely depends on internal sulfated domains that mediate specific binding to an extensive range of proteins. In this study we employed anion exchange chromatography, molecular sieving and enzymatic cleavage on HS fractions purified from three compartments of cultured osteoblasts-soluble conditioned media, cell surface, and extracellular matrix (ECM). We demonstrate that the composition of HS chains purified from the different compartments is structurally non-identical by a number of parameters, and that these differences have significant ramifications for their ligand-binding properties. The HS chains purified of conditioned medium had twice the binding affinity for FGF2 when compared with either cell surface or ECM HS. In contrast, similar binding of BMP2 to the three types of HS was observed. These results suggest that different biological compartments of cultured cells have structurally and functionally distinct HS species that help to modulate the flow of HS-dependent factors between the ECM and the cell surface.

The effect of lumbar support on the trunk muscle activity and pelvic tilt in a passenger vehicle car seat

In order to gain a competitive edge in the market, automotive manufacturers and automotive seat suppliers have identified seat ergonomics for further development to improve overall vehicle comfort. Adjustable lumbar support devices have been offered since long as comfort systems in either a 2-way or 4-way adjustable configuration, although their effect on lumbar strain is not well documented. The effect of a lumbar support on posture and muscular strain, and therefore the relationship between discomfort and comfort device parameter settings, requires clarification. The aim of this paper is to study the effect of a 4-way lumbar support on lower trunk and pelvis muscle activity, pelvic tilt and spine curvature during a car seating activity. 10 healthy subjects (5 m/f; age 19-39) performed a seating activity in a passenger vehicle with seven different static lumbar support positions. The lumbar support was tested in 3 different height positions in relation to the seatback surface centreline (high, centre, low), each having 2 depths positions (lumbar prominence). An extra depth position was added for the centre position. Posture data were collected using a VICON MX motion capture system and NORAXON DTS goniometers and inclinometer. A rigid-body model of an adjustable car seat with four-way adjustable lumbar support was constructed in UGS Siemens NX and connected to a musculoskeletal model of a seated-human, modelled in AnyBody. Wireless electromyography (EMG) was used to calibrate the musculoskeletal model and assess the relationship between (a) muscular strain and lumbar prominence (normal to seatback surface) respective to the lumbar height (alongside seatback surface), (b) hip joint moment and lumbar prominence (normal to seatback surface) respective to lumbar height (alongside seatback surface) and (c) pelvic tilt and lumbar prominence (normal to seatback surface) respective to the lumbar height (alongside seatback surface). This study was based on the assumption that the musculoskeletal human model was seated at the correct R-Point (SgRP), determined via the occupant packaging toolkit in the JACK digital human model. The effect of the interaction between the driver/car-seat has been investigated for factors resulting from the presence and adjustment of a 4-way lumbar support. The results obtained show that various seat adjustments, and driver’s lumbar supports can have complex influence on the muscle activation, joint forces and moments, all of which can affect the comfort perception of the driver. This study enables the automotive industry to optimise passenger vehicle seat development and design. It further more supports the evaluation of static postural and dynamic seat comfort in normal everyday driving tasks and can be applied for future car design to reduce investment and improve comfort.

Digital human modelling for vehicle design and manufacturing

Digital human modelling (DHM) has today matured from research into industrial application. In the automotive domain, DHM has become a commonly used tool in virtual prototyping and human-centred product design. While this generation of DHM supports the ergonomic evaluation of new vehicle design during early design stages of the product, by modelling anthropometry, posture, motion or predicting discomfort, the future of DHM will be dominated by CAE methods, realistic 3D design, and musculoskeletal and soft tissue modelling down to the micro-scale of molecular activity within single muscle fibres. As a driving force for DHM development, the automotive industry has traditionally used human models in the manufacturing sector (production ergonomics, e.g. assembly) and the engineering sector (product ergonomics, e.g. safety, packaging). In product ergonomics applications, DHM share many common characteristics, creating a unique subset of DHM. These models are optimised for a seated posture, interface to a vehicle seat through standardised methods and provide linkages to vehicle controls. As a tool, they need to interface with other analytic instruments and integrate into complex CAD/CAE environments. Important aspects of current DHM research are functional analysis, model integration and task simulation. Digital (virtual, analytic) prototypes or digital mock-ups (DMU) provide expanded support for testing and verification and consider task-dependent performance and motion. Beyond rigid body mechanics, soft tissue modelling is evolving to become standard in future DHM. When addressing advanced issues beyond the physical domain, for example anthropometry and biomechanics, modelling of human behaviours and skills is also integrated into DHM. Latest developments include a more comprehensive approach through implementing perceptual, cognitive and performance models, representing human behaviour on a non-physiologic level. Through integration of algorithms from the artificial intelligence domain, a vision of the virtual human is emerging.

Deep Raman spectroscopy for the non-invasive standoff detection of concealed chemical threat agents

Deep Raman spectroscopy has been utilized for the standoff detection of concealed chemical threat agents from a distance of 15 meters under real life background illumination conditions. By using combined time and space resolved measurements, various explosive precursors hidden in opaque plastic containers were identified non-invasively. Our results confirm that combined time and space resolved Raman spectroscopy leads to higher selectivity towards the sub-layer over the surface layer as well as enhanced rejection of fluorescence from the container surface when compared to standoff spatially offset Raman spectroscopy. Raman spectra that have minimal interference from the packaging material and good signal-to-noise ratio were acquired within 5 seconds of measurement time. A new combined time and space resolved Raman spectrometer has been designed with nanosecond laser excitation and gated detection, making it of lower cost and complexity than picosecond-based laboratory systems.

The contribution of seat components to seat hardness and the interface between human occupant and a driver seat

Effective digital human model (DHM) simulation of automotive driver packaging ergonomics, safety and comfort depends on accurate modelling of occupant posture, which is strongly related to the mechanical interaction between human body soft tissue and flexible seat components. This paper comprises: a study investigating the component mechanical behaviour of a spring-suspended, production level seat when indented by SAE J826 type, human thigh-buttock representing hard shell; a model of seated human buttock shape for improved indenter design using a multivariate representation of Australian population thigh-buttock anthropometry; and a finite-element study simulating the deflection of human buttock and thigh soft tissue when seated, based on seated MRI. The results of the three studies provide a description of the mechanical properties of the driver-seat interface, and allow validation of future dynamic simulations, involving multi-body and finite-element (FE) DHM in virtual ergonomic studies.

Books, toys and tablets : playing and learning in the age of digital media

Introduction During a recent study of how parents source information about children‘s early learning, one of us made our first serious foray into a local store licensed to the global chain Toys'R' Us. While walking the aisles, closely observing layout, signage and stock, several things became obvious. Firstly, large numbers of toys were labeled'educational'. Secondly, many toys in that category were intended for children under the age of two years. These were further differentiated as intended for 'babies' or 'infants', and sub-categorized on packaging or shelving using even smaller age increments (e.g. 0-3 months, 12-18 months, and so on). Thirdly, many products were labeled as 'interactive' and 'learning' toys that promised to assist children‘s early learning and development. The activation of some of these toys relied on embedded computer chip technology and promised to 'connect' children with the home television, computer and the Internet. These products were hybrids between a toy and a platform for digital media interaction. Closer inspection of toy packaging and other promotional material suggested that industry had begun to invest heavily in developing highly differentiated children‘s markets for products that yoked together concepts of learning and development, the 'fun toy' that incorporates digital technology, and offline- and online participation. In this chapter we explore the growth of this contemporary cultural phenomenon that now connects books, toys and mobile digital media with children‘s play and learning.

A research framework for assessing the effects of heat stress on construction workers

Hong Kong in summer (June - October) is hot and humid. Construction workers have to undertake physically demanding activities and often in confined spaces. They are vulnerable to heat stress in summer hence health and safety measures associated to heat stress measured by scientific and clinical parameters are urgently needed. This paper provides an initial report of a research project funded by the Research Grants Council (RGC) of the HKSAR. The aim of this study is to develop a set of indices measured by clinical and scientific methods to detect impending attacks of heat stress. These indices would be of tremendous value in better safeguarding workers’ health and safety by reducing the occurrences of heat stress on site. This paper firstly reports on the statistics of construction incidents arising from heat stress. Qualitative and quantitative research methods applied in conducting the research are discussed. It is believed that the construction industry and the government would benefit a lot as a result of this study.

Real-time detection of concealed chemical hazards under ambient light conditions using Raman spectroscopy

Current concerns regarding terrorism and international crime highlight the need for new techniques for detecting unknown and hazardous substances. A novel Raman spectroscopy-based technique, spatially offset Raman spectroscopy (SORS), was recently devised for non-invasively probing the contents of diffusely scattering and opaque containers. Here, we demonstrate a modified portable SORS sensor for detecting concealed substances in-field under different background lighting conditions. Samples including explosive precursors, drugs and an organophosphate insecticide (chemical warfare agent surrogate) were concealed inside diffusely scattering packaging including plastic, paper and cloth. Measurements were carried out under incandescent and fluorescent light as well as under daylight to assess the suitability of the probe for different real-life conditions. In each case, it was possible to identify the substances against their reference Raman spectra in less than one minute. The developed sensor has potential for rapid detection of concealed hazardous substances in airports, mail distribution centers and customs checkpoints.

Development of a 3D culture system to study the skeletal metastasis of prostate cancer

In the cancer research field, most in vitro studies still rely on two-dimensional (2D) cultures. However, the trend is rapidly shifting towards using a three-dimensional (3D) culture system. This is because 3D models better recapitulate the microenvironment of cells, and therefore, yield cellular and molecular responses that more accurately describe the pathophysiology of cancer. By adopting technology platforms established by the tissue engineering discipline, it is now possible to grow cancer cells in extracellular matrix (ECM)-like environments and dictate the biophysical and biochemical properties of the matrix. In addition, 3D models can be modified to recapitulate different stages of cancer progression for instance from the initial development of tumor to metastasis. Inevitably, to recapitulate a heterotypic condition, comprising more than one cell type, it requires a more complex 3D model. To date, 3D models that are available for studying the prostate cancer (CaP)-bone interactions are still lacking. Therefore, the aim of this study is to establish a co-culture model that allows investigation of direct and indirect CaP-bone interactions. Prior to that, 3D polyethylene glycol (PEG)-based hydrogel cultures for CaP cells were first developed and growth conditions were optimised. Characterization of the 3D hydrogel cultures show that LNCaP cells form a multicellular mass that resembles avascular tumor. In comparison to 2D cultures, besides the difference in cell morphology, the response of LNCaP cells to the androgen analogue (R1881) stimulation is different compared to the cells in 2D cultures. This discrepancy between 2D and 3D cultures is likely associated with the cell-cell contact, density and ligand-receptor interactions. Following the 3D monoculture study, a 3D direct co-culture model of CaP cells and the human tissue engineered bone (hTEBC) construct was developed. Interactions between the CaP cells and human osteoblasts (hOBs) resulted in elevation of Matrix Metalloproteinase 9 (MMP9) for PC-3 cells and Prostate Specific Antigen (PSA) for LNCaP cells. To further investigate the paracrine interaction of CaP cells and (hOBs), a 3D indirect co-culture model was developed, where LNCaP cells embedded within PEG hydrogels were co-cultured with hTEBC. It was found that the cellular changes observed reflect the early event of CaP colonizing the bone site. In the absence of androgens, interestingly, up-regulation of PSA and other kallikreins is also detected in the co-culture compared to the LNCaP monoculture. This non androgenic stimulation could be triggered by the soluble factors secreted by the hOB such as Interleukin-6. There are also decrease in alkaline phosphatase (ALP) activity and down-regulation of genes of the hOB when co-cultured with LNCaP cells that have not been previously described. These genes include transforming growth factor β1 (TGFβ1), osteocalcin and Vimentin. However, no changes to epithelial markers (e.g E-cadherin, Cytokeratin 8) were observed in both cell types from the co-culture. Some of these intriguing changes observed in the co-cultures that had not been previously described have enriched the basic knowledge of the CaP cell-bone interaction. From this study, we have shown evidence of the feasibility and versatility of our established 3D models. These models can be adapted to test various hypotheses for studies pertaining to underlying mechanisms of bone metastasis and could provide a vehicle for anticancer drug screening purposes in the future.

Fast fourier analysis of structural organization in decellularized cartilage-on-bone laminates

Denaturation of tissues can provide a unique biological environment for regenerative medicine application only if minimal disruption of their microarchitecture is achieved during the decellularization process. The goal is to keep the structural integrity of such a construct as functional as the tissues from which they were derived. In this work, cartilage-on-bone laminates were decellularized through enzymatic, non-ionic and ionic protocols. This work investigated the effects of decellularization process on the microarchitecture of cartiligous extracellular matrix; determining the extent of how each process deteriorated the structural organization of the network. High resolution microscopy was used to capture cross-sectional images of samples prior to and after treatment. The variation of the microarchitecture was then analysed using a well defined fast Fourier image processing algorithm. Statistical analysis of the results revealed how significant the alternations among aforementioned protocols were (p < 0.05). Ranking the treatments by their effectiveness in disrupting the ECM integrity, they were ordered as: Trypsin> SDS> Triton X-100.

An examination of recent taxation amendments, corporate law reforms and Queensland legislative reforms affecting nonprofit organisations

There is a tax amendment bill which will be debated. The Government has promised to outline its plan for the reform of the taxation system sometime this year. The plans appear to go beyond the mere introduction of some sort of goods and services tax to reform of the whole taxation system including fiscal relations with the States. Not for profit organisations will find their taxation environment will change. Governments are reluctant to permit exemptions to a GST style arrangements. GST trade offs such as reduced income tax rates and abolishing indirect taxes are useless to nonprofit organisations, as many are already exempt from such imposts. Administrative changes to tax collections may also have an impact. If the government decides to make an individual PAYE taxpayer return optional in exchange for no or standard deductions, this may have an effect on fundraising. The FBT and salary packaging schemes that not for profit organisations use will be under intense scrutiny. A regionalisation of the ATO along the successful model of the ASC would see discrete areas such as not for profit exemptions being centralised in one regional office for the whole of Australia. For example the Tasmanian ASC Office has the responsibility for much work in respect of corporate charities and not for profit companies.

Health matters for subscribers to community-supported agriculture

There are a variety of reasons and motivations for people to subscribe to community-supported agriculture (CSA) schemes, many of which include social, ethical, environmental, and economical benefits. The global rise of food allergies and food related health issues in recent years has led to a growing number of initiatives particularly in developing countries to raise more awareness of the current situation amongst individuals, organisations, and government bodies, and to plan for its implications for the existing food and health systems. Based on a mixed method research conducted in Australia, this paper argues that personal health matters are one of the key motivators for consumers to seek out alternative food systems, particularly CSA initiatives. In addition, it presents the willingness for consumers to seek out information about the food they consume and proposes that technology plays a key role in being used as a conduit to share and investigate information relating to alternative food systems. Further research is required to determine the variety of benefits and opportunities alternative food systems can provide consumers with food related health issues.