The osteogenic differentiation of adipose tissue-derived precursor cells in A 3D scaffold/matrix environment

Abstract: This paper details an in-vitro study using human adipose tissue-derived precursor/stem cells (ADSCs) in three-dimensional (3D) tissue culture systems. ADSCs from 3 donors were seeded onto NaOH-treated medical grade polycaprolactone-tricalcium phosphate (mPCL-TCP) scaffolds with two different matrix components; fibrin glue and lyophilized collagen. ADSCs within these scaffolds were then induced to differentiate along the osteogenic lineage for a 28-day period and various assays and imaging techniques were performed at Day 1, 7, 14, 21 and 28 to assess and compare the ADSC’s adhesion, viability, proliferation, metabolism and differentiation along the osteogenic lineage when cultured in the different scaffold/matrix systems. The ADSC cells were proliferative in both collagen and fibrin mPCL-TCP scaffold systems with a consistently higher cell number (by comparing DNA amounts) in the induced group over the non-induced groups for both scaffold systems. In response to osteogenic induction, these ADSCs expressed elevated osteocalcin, alkaline phosphatase and osteonectin levels. Cells were able to proliferate within the pores of the scaffolds and form dense cellular networks after 28 days of culture and induction. The successful cultivation of osteogenic by FDM process manufactured ADSCs within a 3D matrix comprising fibrin glue or collagen, immobilized within a robust synthetic scaffold is a promising technique which should enhance their potential usage in the regenerative medicine arena, such as bone tissue engineering.

Driver behaviour and decision making at railway level crossings : An exploratory on-road case study

Crashes at rail level crossings represent a significant problem, both in Australia and worldwide. Advances in driving assessment methods, such as the provision of on-road instrumented test vehicles, now provide researchers with the opportunity to further understand driver behaviour at rail level crossings in ways not previously possible. This paper gives an overview of a recent on-road pilot study of driver behaviour at rail level crossings in which 25 participants drove a pre-determined route, incorporating 4 rail level crossings, using MUARC's instrumented On-Road Test Vehicle (ORTeV). Drivers provided verbal commentary whilst driving the route, and a range of other data were collected, including eye fixations, forward, cockpit and driver video, and vehicle data (speed, braking, steering wheel angle, lane tracking etc). Participants also completed a post trial cognitive task analysis interview. Extracts from the wider analyses are used to examine in depth driver behaviour at one of the rail level crossings encountered during the study. The analysis presented, along with the overall analysis undertaken, gives insight into the driver and wider systems factors that shape behaviour at rail level crossings, and highlights the utility of using a multi-method, instrumented vehicle approach for gathering data regarding driver behaviour in different contexts.

Comprehensive evaluation of driver gaze pattern using fuzzy rules for driver training

The over representation of novice drivers in crashes is alarming. Research indicates that one in five drivers’ crashes within their first year of driving. Driver training is one of the interventions aimed at decreasing the number of crashes that involve young drivers. Currently, there is a need to develop comprehensive driver evaluation system that benefits from the advances in Driver Assistance Systems. Since driving is dependent on fuzzy inputs from the driver (i.e. approximate distance calculation from the other vehicles, approximate assumption of the other vehicle speed), it is necessary that the evaluation system is based on criteria and rules that handles uncertain and fuzzy characteristics of the drive. This paper presents a system that evaluates the data stream acquired from multiple in-vehicle sensors (acquired from Driver Vehicle Environment-DVE) using fuzzy rules and classifies the driving manoeuvres (i.e. overtake, lane change and turn) as low risk or high risk. The fuzzy rules use parameters such as following distance, frequency of mirror checks, gaze depth and scan area, distance with respect to lanes and excessive acceleration or braking during the manoeuvre to assess risk. The fuzzy rules to estimate risk are designed after analysing the selected driving manoeuvres performed by driver trainers. This paper focuses mainly on the difference in gaze pattern for experienced and novice drivers during the selected manoeuvres. Using this system, trainers of novice drivers would be able to empirically evaluate and give feedback to the novice drivers regarding their driving behaviour.

Dynamics of in vitro polymer degradation of polycaprolactone-based scaffolds : accelerated versus simulated physiological conditions

The increasing use of biodegradable devices in tissue engineering and regenerative medicine means it is essential to study and understand their degradation behaviour. Accelerated degradation systems aim to achieve similar degradation profiles within a shorter period of time, compared with standard conditions. However, these conditions only partially mimic the actual situation, and subsequent analyses and derived mechanisms must be treated with caution and should always be supported by actual long-term degradation data obtained under physiological conditions. Our studies revealed that polycaprolactone (PCL) and PCL-composite scaffolds degrade very differently under these different degradation conditions, whilst still undergoing hydrolysis. Molecular weight and mass loss results differ due to the different degradation pathways followed (surface degradation pathway for accelerated conditions and bulk degradation pathway for simulated physiological conditions). Crystallinity studies revealed similar patterns of recrystallization dynamics, and mechanical data indicated that the scaffolds retained their functional stability, in both instances, over the course of degradation. Ultimately, polymer degradation was shown to be chiefly governed by molecular weight, crystallinity susceptibility to hydrolysis and device architecture considerations whilst maintaining its thermodynamic equilibrium.

In vitro cultivation of adult human chondrocytes : importance of culture system, oxygen and zonal differences

Articular cartilage exhibits limited intrinsic regenerative capacity and focal tissue defects can lead to the development of osteoarthritis (OA), a painful and debilitating loss of cartilage tissue. In Australia, 1.4 million people are affected by OA and its prevalence is increasing in line with current demographics. As treatment options are limited, new therapeutic approaches are being investigated including biological resurfacing of joints with tissue-engineered cartilage. Despite some progress in the field, major challenges remain to be addressed for large scale clinical success. For example, large numbers of chondrogenic cells are required for cartilage formation, but chondrocytes lose their chondrogenic phenotype (dedifferentiate) during in vitro propagation. Additionally, the zonal organization of articular cartilage is critical for normal cartilage function, but development of zonal structure has been largely neglected in cartilage repair strategies. Therefore, we hypothesised that culture conditions for freshly isolated human articular chondrocytes from non-OA and OA sources can be improved by employing microcarrier cultures and a reduced oxygen environment and that oxygen is a critical factor in the maintenance of the zonal chondrocyte phenotype. Microcarriers have successfully been used to cultivate bovine chondrocytes, and offer a potential alternative for clinical expansion of human chondrocytes. We hypothesised that improved yields can be achieved by propagating human chondrocytes on microcarriers. We found that cells on microcarriers acquired a flattened, polygonal morphology and initially proliferated faster than monolayercultivated cells. However, microcarrier cultivation over four weeks did not improve growth rates or the chondrogenic potential of non-OA and OA human articular chondrocytes over conventional monolayer cultivation. Based on these observations, we aimed to optimise culture conditions by modifying oxygen tension, to more closely reflect the in vivo environment. We found that propagation at 5% oxygen tension (moderate hypoxia) did not improve proliferation or redifferentiation capacity of human osteoarthritic chondrocytes. Moderate hypoxia increased the expression of chondrogenic markers during redifferentiation. However, osteoarthritic chondrocytes cultivated on microcarriers exhibited lower expression levels of chondrogenic surface marker proteins and had at best equivalent redifferentiation capacities compared to monolayer-cultured cells. This suggests that monolayer culture with multiple passaging potentially selects for a subpopulation of cells with higher differentiation capacity, which are otherwise rare in osteoarthritic, aged cartilage. However, fibroblastic proteins were found to be highly expressed in all cultures of human osteoarthritic chondrocytes indicating the presence of a high proportion of dedifferentiated, senescent cells with a chondrocytic phenotype that was not rescued by moderate hypoxia. The different zones of cartilage support chondrocyte subpopulations, which exhibit characteristic protein expression and experience varying oxygen tensions. We, therefore, hypothesised that oxygen tension affects the zonal marker expression of human articular chondrocytes isolated from the different cartilage layers. We found that zonal chondrocytes maintained these phenotypic differences during in vitro cultivation. Low oxygen environments favoured the expression of the zonal marker proteoglycan 4 in superficial cells, most likely through the promotion of chondrogenesis. The putative zonal markers clusterin and cartilage intermediate layer protein were found to be expressed by all subpopulations of human osteoarthritic chondrocytes ex vivo and, thus, may not be reliable predictors of in vitro stratification using these clinically relevant cells. The findings in this thesis underline the importance of considering low oxygen conditions and zonal stratification when creating native-like cartilaginous constructs. We have not yet found the right cues to successfully cultivate clinically-relevant human osteoarthritic chondrocytes in vitro. A more thorough understanding of chondrocyte biology and the processes of chondrogenesis are required to ensure the clinical success of cartilage tissue engineering.

Testing of a distributed generation system with a virtual grid

The paper proposes a solution for testing of a physical distributed generation system (DGs) along with a computer simulated network. The computer simulated network is referred as the virtual grid in this paper. Integration of DG with the virtual grid provides broad area of testing of power supplying capability and dynamic performance of a DG. It is shown that a DG can supply a part of load power while keeping Point of Common Coupling (PCC) voltage magnitude constant. To represent the actual load, a universal load along with power regenerative capability is designed with the help of voltage source converter (VSC) that mimics the load characteristic. The overall performance of the proposed scheme is verified using computer simulation studies.

To err (on the road) is human? An on-road study of driver errors

Human error, its causes and consequences, and the ways in which it can be prevented, remain of great interest to road safety practitioners. This paper presents the findings derived from an on-road study of driver errors in which 25 participants drove a pre-determined route using MUARC's On-Road Test Vehicle (ORTeV). In-vehicle observers recorded the different errors made, and a range of other data was collected, including driver verbal protocols, forward, cockpit and driver video, and vehicle data (speed, braking, steering wheel angle, lane tracking etc). Participants also completed a post trial cognitive task analysis interview. The drivers tested made a range of different errors, with speeding violations, both intentional and unintentional, being the most common. Further more detailed analysis of a sub-set of specific error types indicates that driver errors have various causes, including failures in the wider road 'system' such as poor roadway design, infrastructure failures and unclear road rules. In closing, a range of potential error prevention strategies, including intelligent speed adaptation and road infrastructure design, are discussed.

Clonal characterization of bone marrow derived stem cells and their application for bone regeneration

Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However, all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single- cell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homo- geneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous popu- lation. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in vivo

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing : Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro μCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (pb0.01). Younger animals showed (i) larger callus strut thickness (pb0.001), (ii) lower perforation in struts (pb0.01), and (iii) higher mineralization of callus struts (pb0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (pb0.05). Blood vessel density was reduced in animals with semirigid fixation (pb0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca2+ signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells

The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell-matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs (e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases (e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40-60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.

The conflict resolution in connected railway junctions

Purpose of study: Traffic conflicts occur when trains on different routes approach a converging junction in a railway network at the same time. To prevent collisions, a right-of-way assignment is needed to control the order in which the trains should pass the junction. Such control action inevitably requires the braking and/or stopping of trains, which lengthens their travelling times and leads to delays. Train delays cause a loss of punctuality and hence directly affect the quality of service. It is therefore important to minimise the delays by devising a suitable right-of-way assignment. One of the major difficulties in attaining the optimal right-of-way assignment is that the number of feasible assignments increases dramatically with the number of trains. Connected-junctions further complicate the problem. Exhaustive search for the optimal solution is time-consuming and infeasible for area control (multi-junction). Even with the more intelligent deterministic optimisation method revealed in [1], the computation demand is still considerable, which hinders real-time control. In practice, as suggested in [2], the optimality may be traded off by shorter computation time, and heuristic searches provide alternatives for this optimisation problem.

A review of simulation models for railway systems

With the advances in computer hardware and software development techniques in the past 25 years, digital computer simulation of train movement and traction systems has been widely adopted as a standard computer-aided engineering tool [1] during the design and development stages of existing and new railway systems. Simulators of different approaches and scales are used extensively to investigate various kinds of system studies. Simulation is now proven to be the cheapest means to carry out performance predication and system behaviour characterisation. When computers were first used to study railway systems, they were mainly employed to perform repetitive but time-consuming computational tasks, such as matrix manipulations for power network solution and exhaustive searches for optimal braking trajectories. With only simple high-level programming languages available at the time, full advantage of the computing hardware could not be taken. Hence, structured simulations of the whole railway system were not very common. Most applications focused on isolated parts of the railway system. It is more appropriate to regard those applications as primarily mechanised calculations rather than simulations. However, a railway system consists of a number of subsystems, such as train movement, power supply and traction drives, which inevitably contains many complexities and diversities. These subsystems interact frequently with each other while the trains are moving; and they have their special features in different railway systems. To further complicate the simulation requirements, constraints like track geometry, speed restrictions and friction have to be considered, not to mention possible non-linearities and uncertainties in the system. In order to provide a comprehensive and accurate account of system behaviour through simulation, a large amount of data has to be organised systematically to ensure easy access and efficient representation; the interactions and relationships among the subsystems should be defined explicitly. These requirements call for sophisticated and effective simulation models for each component of the system. The software development techniques available nowadays allow the evolution of such simulation models. Not only can the applicability of the simulators be largely enhanced by advanced software design, maintainability and modularity for easy understanding and further development, and portability for various hardware platforms are also encouraged. The objective of this paper is to review the development of a number of approaches to simulation models. Attention is, in particular, given to models for train movement, power supply systems and traction drives. These models have been successfully used to enable various ‘what-if’ issues to be resolved effectively in a wide range of applications, such as speed profiles, energy consumption, run times etc.

Fumaric acid monoethyl ester-functionalized poly(D,L-Lactide)/N-vinyl-2- pyrrolidone Resins for the preparation of tissue engineering scaffolds by stereolithography

Polymer networks were prepared by photocross-linking fumaric acid monoethyl ester (FAME) functionalized, three-armed poly(D,L-lactide) oligomers using Af-vinyl-2-pyrrolidone (NVP) as diluent and comonomer. The use of NVP together with FAME-functionalized oligomers resulted in copolymerization at high rates, and networks with gel contents in excess of 90 were obtained. The hydrophilicity of the poly(D,L-lactide) networks increases with increasing amounts of NVP, networks containing 50 wt of NVP absorbed 40 of water. As the amount of NVP was increased from 30 to 50 wt , the Young's modulus after equilibration in water decreased from 0.8 to 0.2 GPa, as opposed to an increase from 1.5 to 2.1 GPa in the dry state. Mouse preosteoblasts readily adhered and spread onto all prepared networks. Using stereolithography, porous structures with a well-defined gyroid architecture were prepared from these novel materials. This allows the preparation of tissue engineering scaffolds with optimized pore architecture and tunable material properties.