Development of zonal cartilage constructs : effects of chondrocyte subpopulation, compressive stimulation, and culture models

Articular cartilage is a highly resilient tissue located at the ends of long bones. It has a zonal structure, which has functional significance in load-bearing. Cartilage does not spontaneously heal itself when damaged, and untreated cartilage lesions or age-related wear often lead to osteoarthritis (OA). OA is a degenerative condition that is highly prevalent, age-associated, and significantly affects patient mobility and quality of life. There is no cure for OA, and patients usually resort to replacing the biological joint with an artificial prosthesis. An alternative approach is to dynamically regenerate damaged or diseased cartilage through cartilage tissue engineering, where cells, materials, and stimuli are combined to form new cartilage. However, despite extensive research, major limitations remain that have prevented the wide-spread application of tissue-engineered cartilage. Critically, there is a dearth of information on whether autologous chondrocytes obtained from OA patients can be used to successfully generate cartilage tissues with structural hierarchy typically found in normal articular cartilage. I aim to address these limitations in this thesis by showing that chondrocyte subpopulations isolated from macroscopically normal areas of the cartilage can be used to engineer stratified cartilage tissues and that compressive loading plays an important role in zone-dependent biosynthesis of these chondrocytes. I first demonstrate that chondrocyte subpopulations from the superficial (S) and middle/deep (MD) zones of OA cartilage are responsive to compressive stimulation in vitro, and that the effect of compression on construct quality is zone-dependent. I also show that compressive stimulation can influence pericelluar matrix production, matrix metalloproteinase secretion, and cytokine expression in zonal chondrocytes in an alginate hydrogel model. Subsequently, I focus on recreating the zonal structure by forming layered constructs using the alginate-released chondrocyte (ARC) method either with or without polymeric scaffolds. Resulting zonal ARC constructs had hyaline morphology, and expressed cartilage matrix molecules such as proteoglycans and collagen type II in both scaffold-free and scaffold-based approaches. Overall, my findings demonstrate that chondrocyte subpopulations obtained from OA joints respond sensitively to compressive stimulation, and are able to form cartilaginous constructs with stratified organization similar to native cartilage using the scaffold-free and scaffold-based ARC technique. The ultimate goal in tissue engineering is to help provide improved treatment options for patients suffering from debilitating conditions such as OA. Further investigations in developing functional cartilage replacement tissues using autologous chondrocytes will bring us a step closer to improving the quality of life for millions of OA patients worldwide.

Piecing together an integrative taxonomic puzzle: microsatellite, wing shape and aedeagus length analyses of Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in a proposed contact zone along the Thai/Malay Peninsula

Bactrocera dorsalis (Hendel) and B. papayae Drew & Hancock represent a closely related sibling species pair for which the biological species limits are unclear; i.e., it is uncertain if they are truely two biological species, or one biological species which has been incorrectly taxonomically split. The geographic ranges of the two taxa are thought to abut or overlap on or around the Isthmus of Kra, a recognised biogeographic barrier located on the narrowest portion of the Thai Peninsula. We collected fresh material of B. dorsalis sensu lato (i.e., B. dorsalis sensu stricto + B. papayae) in a north-south transect down the Thai Peninsula, from areas regarded as being exclusively B. dorsalis s.s., across the Kra Isthmus, and into regions regarded as exclusively B. papayae. We carried out microsatellite analyses and took measurements of male genitalia and wing shape. Both the latter morphological tests have been used previously to separate these two taxa. No significant population structuring was found in the microsatellite analysis and results were consistent with an interpretation of one, predominantly panmictic population. Both morphological datasets showed consistent, clinal variation along the transect, with no evidence for disjunction. No evidence in any tests supported historical vicariance driven by the Isthmus of Kra, and none of the three datasets supported the current taxonomy of two species. Rather, within and across the area of range overlap or abutment between the two species, only continuous morphological and genetic variation was recorded. Recognition that morphological traits previously used to separate these taxa are continuous, and that there is no genetic evidence for population segregation in the region of suspected species overlap, is consistent with a growing body of literature that reports no evidence of biological differentiation between these taxa.

The "free-factor" impact on value in health services

Purpose - This paper seeks to understand the impact of financial cost on customer value in health prevention services by comparing free government services with private fee-charging providers. This is important as there is a common belief that a free health service is of lower quality and thus lower value than a paid service. However there is no evidence to verify this notion. Design / Methodology / Approach - A large-scale online survey was administered nationwide to Australian women. The respondents were asked about the functional and emotional value derived from their service experiences. Findings - Structural equation modelling (SEM) revealed non significant relationships between fee/free services and functional and emotional value (FV/EV). The non-significant relationship with FV is contrary to the theory of price quality relationship in services. This could be attributed to consumer perceptions that the technical quality of health professionals is comparable across free and paid services. The non-significant relationship with EV could be explained by the indicators used to reflect EV. These indicators were reflective of breast screening behaviour, not breast screening services. Subsequently, it may be posited that the act of having a breast screen is sufficient for consumers to derive emotional value, regardless of the financial cost. Originality / Value - This research fills an important gap in the literature by investigating the impact of financial cost on a service that consumers use proactively(prevention), rather than reactively (treatment). Insights are provided into the impact of cost on customer value in preventive health services, which are valuable to social marketing academics, health practitioners, and governments

Pedigree-free animal models : the relatedness matrix reloaded

Animal models typically require a known genetic pedigree to estimate quantitative genetic parameters. Here we test whether animal models can alternatively be based on estimates of relatedness derived entirely from molecular marker data. Our case study is the morphology of a wild bird population, for which we report estimates of the genetic variance-covariance matrices (G) of six morphological traits using three methods: the traditional animal model; a molecular marker-based approach to estimate heritability based on Ritland's pairwise regression method; and a new approach using a molecular genealogy arranged in a relatedness matrix (R) to replace the pedigree in an animal model. Using the traditional animal model, we found significant genetic variance for all six traits and positive genetic covariance among traits. The pairwise regression method did not return reliable estimates of quantitative genetic parameters in this population, with estimates of genetic variance and covariance typically being very small or negative. In contrast, we found mixed evidence for the use of the pedigree-free animal model. Similar to the pairwise regression method, the pedigree-free approach performed poorly when the full-rank R matrix based on the molecular genealogy was employed. However, performance improved substantially when we reduced the dimensionality of the R matrix in order to maximize the signal to noise ratio. Using reduced-rank R matrices generated estimates of genetic variance that were much closer to those from the traditional model. Nevertheless, this method was less reliable at estimating covariances, which were often estimated to be negative. Taken together, these results suggest that pedigree-free animal models can recover quantitative genetic information, although the signal remains relatively weak. It remains to be determined whether this problem can be overcome by the use of a more powerful battery of molecular markers and improved methods for reconstructing genealogies.

A variable-stepsize Jacobian-free exponential integrator for simulating transport in heterogeneous porous media : application to wood drying

A Jacobian-free variable-stepsize method is developed for the numerical integration of the large, stiff systems of differential equations encountered when simulating transport in heterogeneous porous media. Our method utilises the exponential Rosenbrock-Euler method, which is explicit in nature and requires a matrix-vector product involving the exponential of the Jacobian matrix at each step of the integration process. These products can be approximated using Krylov subspace methods, which permit a large integration stepsize to be utilised without having to precondition the iterations. This means that our method is truly "Jacobian-free" - the Jacobian need never be formed or factored during the simulation. We assess the performance of the new algorithm for simulating the drying of softwood. Numerical experiments conducted for both low and high temperature drying demonstrates that the new approach outperforms (in terms of accuracy and efficiency) existing simulation codes that utilise the backward Euler method via a preconditioned Newton-Krylov strategy.

Comparing measures of fat-free mass in overweight older adults using three different bioelectrical impedance devices and three prediction equations

Objectives: To compare measures of fat-free mass (FFM) by three different bioelectrical impedance analysis (BIA) devices and to assess the agreement between three different equations validated in older adult and/or overweight populations. Design: Cross-sectional study. Setting: Orthopaedics ward of Brisbane public hospital, Australia. Participants: Twenty-two overweight, older Australians (72 yr ± 6.4, BMI 34 kg/m2 ± 5.5) with knee osteoarthritis. Measurements: Body composition was measured using three BIA devices: Tanita 300-GS (foot-to-foot), Impedimed DF50 (hand-to-foot) and Impedimed SFB7 (bioelectrical impedance spectroscopy (BIS)). Three equations for predicting FFM were selected based on their ability to be applied to an older adult and/ or overweight population. Impedance values were extracted from the hand-to-foot BIA device and included in the equations to estimate FFM. Results: The mean FFM measured by BIS (57.6 kg ± 9.1) differed significantly from those measured by foot-to-foot (54.6 kg ± 8.7) and hand-to-foot BIA (53.2 kg ± 10.5) (P < 0.001). The mean ± SD FFM predicted by three equations using raw data from hand-to-foot BIA were 54.7 kg ± 8.9, 54.7 kg ± 7.9 and 52.9 kg ± 11.05 respectively. These results did not differ from the FFM predicted by the hand-to-foot device (F = 2.66, P = 0.118). Conclusions: Our results suggest that foot-to-foot and hand-to-foot BIA may be used interchangeably in overweight older adults at the group level but due to the large limits of agreement may lead to unacceptable error in individuals. There was no difference between the three prediction equations however these results should be confirmed within a larger sample and against a reference standard.