Long-term effects of hydrogel properties on human chondrocyte behavior

Hydrogels provide a 3-dimensional network for embedded cells and offer promise for cartilage tissue engineering applications. Nature-derived hydrogels, including alginate, have been shown to enhance the chondrocyte phenotype but are variable and not entirely controllable. Synthetic hydrogels, including polyethylene glycol (PEG)-based matrices, have the advantage of repeatability and modularity; mechanical stiffness, cell adhesion, and degradability can be altered independently. In this study, we compared the long-term in vitro effects of different hydrogels (alginate and Factor XIIIa-cross-linked MMP-sensitive PEG at two stiffness levels) on the behavior of expanded human chondrocytes and the development of construct properties. Monolayer-expanded human chondrocytes remained viable throughout culture, but morphology varied greatly in different hydrogels. Chondrocytes were characteristically round in alginate but mostly spread in PEG gels at both concentrations. Chondrogenic gene (COL2A1, aggrecan) expression increased in all hydrogels, but alginate constructs had much higher expression levels of these genes (up to 90-fold for COL2A1), as well as proteoglycan 4, a functional marker of the superficial zone. Also, chondrocytes expressed COL1A1 and COL10A1, indicative of de-differentiation and hypertrophy. After 12 weeks, constructs with lower polymer content were stiffer than similar constructs with higher polymer content, with the highest compressive modulus measured in 2.5% PEG gels. Different materials and polymer concentrations have markedly different potency to affect chondrocyte behavior. While synthetic hydrogels offer many advantages over natural materials such as alginate, they must be further optimized to elicit desired chondrocyte responses for use as cartilage models and for development of functional tissue-engineered articular cartilage.

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

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.

Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs

It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labelled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labelled) and S*/M* (both S and M cells labelled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labelled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labelled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.

Depth-dependent biomechanical and biochemical properties of fetal, newborn, and tissue-engineered articular cartilage

Adult articular cartilage has depth-dependent mechanical and biochemical properties which contribute to zone-specific functions. The compressive moduli of immature cartilage and tissue-engineered cartilage are known to be lower than those of adult cartilage. The objective of this study was to determine if such tissues exhibit depth-dependent compressive properties, and how these depth-varying properties were correlated with cell and matrix composition of the tissue. The compressive moduli of fetal and newborn bovine articular cartilage increased with depth (p < 0.05) by a factor of 4-5 from the top 0.1 mm (28 +/- 13 kPa, 141 +/- 10 kPa, respectively) to 1 mm deep into the tissue. Likewise, the glycosaminoglycan and collagen content increased with depth (both p < 0.001), and correlated with the modulus (both p < 0.01). In contrast, tissue-engineered cartilage formed by either layering or mixing cells from the superficial and middle zone of articular cartilage exhibited similarly soft regions at both construct surfaces, as exemplified by large equilibrium strains. The properties of immature cartilage may provide a template for developing tissue-engineered cartilage which aims to repair cartilage defects by recapitulating the natural development and growth processes. These results suggest that while depth-dependent properties may be important to engineer into cartilage constructs, issues other than cell heterogeneity must be addressed to generate such tissues. (c) 2005 Elsevier Ltd. All rights reserved.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

Optimization of container process at seaport terminals

Seaport container terminals are an important part of the logistics systems in international trades. This paper investigates the relationship between quay cranes, yard machines and container storage locations in a multi-berth and multi-ship environment. The aims are to develop a model for improving the operation efficiency of the seaports and to develop an analytical tool for yard operation planning. Due to the fact that the container transfer times are sequence-dependent and with the large number of variables involve, the proposed model cannot be solved in a reasonable time interval for realistically sized problems. For this reason, List Scheduling and Tabu Search algorithms have been developed to solve this formidable and NP-hard scheduling problem. Numerical implementations have been analysed and promising results have been achieved.

Australian Library and Information Studies (LIS) researchers' ranking of LIS journals

The paper describes the processes and the outcomes of the ranking of LIS journal titles by Australia’s LIS researchers during 2007-8, firstly through the Australian federal government’s Research Quality Framework (RQF) process and then its replacement, the Excellence in Research for Australia (ERA) initiative. The requirement to rank the journals titles used came from discussions held at the RQF panel meeting held in February 2007 in Canberra, Australia. While it was recognised that the Web of Science (formerly ISI) journal impact approach of journal acceptance for measures of research quality and impact might not work for LIS, it was apparent that this model would be the default if no other ranking of journal titles became apparent. Although an increasing number of LIS and related discipline journals were appearing in the Web of Science listed rankings, the number was few and it was thus decided by the Australian LIS research community to undertake the ranking exercise.

Profiling off-road motorcycle and all-terrain riders in Queensland, Australia

The current research aimed to profile off-road riders to identify specific sub-groups in relation to their risk-related behaviours and perceptions. A total of 235 adults from the Australian state of Queensland who had ridden a motorcycle or ATV off-road in the last 12 months were recruited. A cluster analysis was applied to the survey data. Two distinct clusters of riders were identified, which corresponded with the self-report of injury from an off-road riding crash in the prior 12 months. The injured cluster had a significantly higher mean risk propensity and use of safety equipment, though did not differ on self-reported risk taking. The injured cluster as a whole included a higher percentage of males, was younger, and rode more often for recreational or competitive purposes than the non-crash involved cluster. The results indicate that the crash cluster may be both more aware of the potential risks of riding and more willing to ride in a riskier manner.

Contemporary children's literature and film : engaging with theory

Bringing together the voices of leading and emerging scholars, this book provides critical approaches for reading children's literature and film. It argues for the significance of theory for reading texts written and produced for young people and integrates a wide range of critical perspectives, including schema theory, theories of space and place, cultural globalization, feminism, ecocriticism, adaptation theory, postcolonialism, and posthumanism.

Deterministic diffraction loss modelling for novel broadband communication in rural environments

This paper presents a deterministic modelling approach to predict diffraction loss for an innovative Multi-User-Single-Antenna (MUSA) MIMO technology, proposed for rural Australian environments. In order to calculate diffraction loss, six receivers have been considered around an access point in a selected rural environment. Generated terrain profiles for six receivers are presented in this paper. Simulation results using classical diffraction models and diffraction theory are also presented by accounting the rural Australian terrain data. Results show that in an area of 900 m by 900 m surrounding the receivers, path loss due to diffraction can range between 5 dB and 35 dB. Diffraction loss maps can contribute to determine the optimal location for receivers of MUSA-MIMO systems in rural areas.

Crystal growth from undercooling melts under strong gradients of temperatures generating in short-duration microgravity

Crystal growth of bulk CdTe in short-duration microgravity is performed by the unidirectional cooling method. The largest growth grains in microgravity samples are 4X2mm. The cooling profiles indicate undercooling melts in microgravity. Cooling melt samples in microgravity generate strong gradient of temperature due to stop thermal convections. Temperature distribution in the melt is calculated by the one-dimensional equation of heat conduction, and about 100 K-undercooling is considered to occur at the cooling surface.