Tissue engineering of stratified articular cartilage from chondrocyte subpopulations

Objective: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. Design: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). Results: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. Conclusions: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Inhibition of integrative cartilage repair by proteoglycan 4 in synovial fluid

To determine the effects of the articular cartilage surface, as well as synovial fluid (SF) and its components, specifically proteoglycan 4 (PRG4) and hyaluronic acid (HA), on integrative cartilage repair in vitro. Methods. Blocks of calf articular cartilage were harvested, some with the articular surface intact and others without. Some of the latter types of blocks were pretreated with trypsin, and then with bovine serum albumin, SF, PRG4, or HA. Immunolocalization of PRG4 on cartilage surfaces was performed after treatment. Pairs of similarly treated cartilage blocks were incubated in partial apposition for 2 weeks in medium supplemented with serum and 3 H-proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength, and protein biosynthesis and deposition were determined by incorporated 3 H-proline. Results. Samples with articular surfaces in apposition exhibited little integrative repair compared with samples with cut surfaces in apposition. PRG4 was immunolocalized at the articular cartilage surface, but not in deeper, cut surfaces (without treatment). Cartilage samples treated with trypsin and then with SF or PRG4 exhibited an inhibition of integrative repair and positive immunostaining for PRG4 at treated surfaces compared with normal cut cartilage samples, while samples treated with HA exhibited neither inhibited integrative repair nor PRG4 at the tissue surfaces. Deposition of newly synthesized protein was relatively similar under conditions in which integration differed significantly. Conclusion. These results support the concept that PRG4 in SF, which normally contributes to cartilage lubrication, can inhibit integrative cartilage repair. This has the desirable effect of preventing fusion of apposing surfaces of articulating cartilage, but has the undesirable effect of inhibiting integrative repair.

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.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.

Relationships between preventive activities, psychosocial factors and recurrence of venous leg ulcers : a prospective study

Aim To identify relationships between preventive activities, psychosocial factors and leg ulcer recurrence in patients with chronic venous leg ulcers. Background Chronic venous leg ulcers are slow to heal and frequently recur, resulting in years of suffering and intensive use of health care resources. Methods A prospective longitudinal study was undertaken with a sample of 80 patients with a venous leg ulcer recruited when their ulcer healed. Data were collected from 2006–2009 from medical records on demographics, medical history and ulcer history; and from self-report questionnaires on physical activity, nutrition, preventive activities and psychosocial measures. Follow-up data were collected via questionnaires every three months for 12 months after healing. Median time to recurrence was calculated using the Kaplan-Meier method. A Cox proportional-hazards regression model was used to adjust for potential confounders and determine effects of preventive strategies and psychosocial factors on recurrence. Results: There were 35 recurrences in a sample of 80 participants. Median time to recurrence was 27 weeks. After adjustment for potential confounders, a Cox proportional hazards regression model found that at least an hour/day of leg elevation, six or more days/week in Class 2 (20–25mmHg) or 3 (30–40mmHg) compression hosiery, higher social support scale scores and higher General Self-Efficacy scores remained significantly associated (p<0.05) with a lower risk of recurrence, while male gender and a history of DVT remained significant risk factors for recurrence. Conclusion Results indicate that leg elevation, compression hosiery, high levels of self-efficacy and strong social support will help prevent recurrence.

Changes in resting and walking energy expenditure and walking speed during pregnancy in obese women

Background Energy conserving processes reported in undernourished women during pregnancy are a recognised strategy to provide energy required to support fetal development. Women who are obese before conceiving arguably have sufficient fat stores to support the energy demands of pregnancy without the need to provoke energy conserving mechanisms. Objective We tested the hypothesis that obese women would demonstrate behavioural adaptation (i.e. decrease in self-selected walking (SSW) speed) but not metabolic compensation (i.e. decrease in resting metabolic rate (RMR) or metabolic cost of walking) during gestation. Design RMR, SSW speed, metabolic cost of walking, and anthropometry were measured in 23 women (BMI: 33.6 ± 2.5 kg/m2; 31 ± 4 years) at approximately weeks 15 (wk 15) and 30 (wk 30) of gestation. RMR was also measured in two cohorts of non-pregnant controls matched for age, weight and height of the pregnant cohort at wk 15 (N=23) and wk 30 (N=23). Results GWG varied widely (11.3 ± 5.4 kg) and 52% of women gained more weight than is recommended. RMR increased significantly by an average 177 ± 176 kcal/d (11±12%; P<0.0001); however the within-group variability was large. Both the metabolic cost of walking and SSW speed decreased significantly (P<0.01). While RMR increased in >80% of the cohort, the net oxygen cost of walking decreased in the same proportion of women. Conclusions While the increase in RMR was greater than was explained by weight gain, there was evidence of both behavioural and biological compensation in the metabolic cost of walking in obese women during gestation.

Maternal exposure to ambient temperature and the risk of preterm birth and stillbirth in Brisbane, Australia

Almost 10% of all births are preterm and 2.2% are stillbirths globally. Recent research has suggested that environmental factors may be a contributory cause to these adverse birth outcomes. The authors examined the relationship between ambient temperature and preterm birth and stillbirth in Brisbane, Australia between 2005 and 2009 (n = 101,870). They used a Cox proportional hazard model with live birth and stillbirth as competing risks. They also examined if there were periods of the pregnancy where exposure to high temperatures had a greater effect. Exposure to higher ambient temperatures during pregnancy increased the risk of stillbirth. The hazard ratio for stillbirth was 0.3 at 12 °C relative to the reference temperature at 21 °C. The temperature effect was greatest for fetuses of less than 36 weeks of gestation. There was an association between higher temperature and shorter gestation, as the hazard ratio for live birth was 0.96 at 15 °C and 1.02 at 25 °C. This effect was greatest at later gestational ages. The results provide strong evidence of an association between increased temperature and increased risk of stillbirth and shorter gestations.

Outcomes and implications of one teacher's approach to context-based science in the middle years

In Australia, there is a crisis in science education with students becoming disengaged with canonical science in the middle years of schooling. One recent initiative that aims to improve student interest and motivation without diminishing conceptual understanding is the context-based approach. Contextual units that connect the canonical science with the students’ real world of their local community have been used in the senior years but are new in the middle years. This ethnographic study explored the learning transactions that occurred in one 9th grade science class studying a context-based Environmental Science unit for 11 weeks. Outcomes of the study and implications are discussed in this paper.

Maximum recovery after knee replacement - the MARKER study rationale and protocol

Background There is little scientific evidence to support the usual practice of providing outpatient rehabilitation to patients undergoing total knee replacement surgery (TKR) immediately after discharge from the orthopaedic ward. It is hypothesised that the lack of clinical benefit is due to the low exercise intensity tolerated at this time, with patients still recovering from the effects of major orthopaedic surgery. The aim of the proposed clinical trial is to investigate the clinical and cost effectiveness of a novel rehabilitation strategy, consisting of an initial home exercise programme followed, approximately six weeks later, by higher intensity outpatient exercise classes. Methods/Design In this multicentre randomised controlled trial, 600 patients undergoing primary TKR will be recruited at the orthopaedic pre-admission clinic of 10 large public and private hospitals in Australia. There will be no change to the medical or rehabilitative care usually provided while the participant is admitted to the orthopaedic ward. After TKR, but prior to discharge from the orthopaedic ward, participants will be randomised to either the novel rehabilitation strategy or usual rehabilitative care as provided by the hospital or recommended by the orthopaedic surgeon. Outcomes assessments will be conducted at baseline (pre-admission clinic) and at 6 weeks, 6 months and 12 months following randomisation. The primary outcomes will be self-reported knee pain and physical function. Secondary outcomes include quality of life and objective measures of physical performance. Health economic data (health sector and community service utilisation, loss of productivity) will be recorded prospectively by participants in a patient diary. This patient cohort will also be followed-up annually for five years for knee pain, physical function and the need or actual incidence of further joint replacement surgery. Discussion The results of this pragmatic clinical trial can be directly implemented into clinical practice. If beneficial, the novel rehabilitation strategy of utilising outpatient exercise classes during a later rehabilitation phase would provide a feasible and potentially cost-effective intervention to optimise the physical well-being of the large number of people undergoing TKR.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

Journalism at the movies

Welcome to the first of what will be a regular review essay on films about journalism, covering recent releases as well as looking back at established classics and under-rated obscurities. And there is plenty to write about. Since 2008, and the end of the research period which informed my 2010 book on Journalists in Film there has been a steady stream of films in which a journalist is a primary character, and in which the nature and functioning of journalism is a theme. Morning Glory (Roger Michell, 2010), the story of a ‘serious’ news man (Harrison Ford) having to adapt to the infotainment environment of breakfast news, came out early in 2011 in the United Kingdom. The well-received UK indie Monsters (Gareth Edwards, 2010), a sci-fi with a journalist at its heart, was released in 2010. In The Soloist (Joe Wright, 2009) Robert Downey Jr played a feature journalist who befriends a mentally ill street musician and seeks to rescue him through his writing. Stieg Larsson’s Millennium trilogy has produced three Swedish films, all of them focused on the campaigning journalist Mikael Blomkvist. The first of these is being remade by Hollywood as of this writing.

A pilot study assessing the feasibility of a home-based progressive resistance exercise program and trend toward healing rates for patients with venous leg ulcers

Venous leg ulceration is a serious condition affecting 1 – 3% of the population. Decline in the function of the calf muscle pump is correlated with venous ulceration. Many previous studies have reported an improvement in the function of the calf muscle pump, endurance of the calf muscle and increased range of ankle motion after structured exercise programs. However, there is a paucity of published research that assesses if these improvements result in an improvement in the healing rates of venous ulcers. The primary purpose of this pilot study was to establish the feasibility of a homebased progressive resistance exercise program and examine if there was any clinical significance or trend toward healing. The secondary aims were to examine the benefit of a home-based progressive resistance exercise program on calf muscle pump function and physical parameters. The methodology used was a randomised controlled trial where eleven participants were randomised into an intervention (n = 6) or control group (n = 5). Participants who were randomised to receive a 12-week home-based progressive resistance exercise program were instructed through weekly face-to-face consultations during their wound clinic appointment by the author. Control group participants received standard wound care and compression therapy. Changes in ulcer parameters were measured fortnightly at the clinic (number healed at 12 weeks, percentage change in area and pressure ulcer score healing score). An air plethysmography test was performed at baseline and following the 12 weeks of training to determine changes in calf muscle pump function. Functional measures included maximum number of heel raises (endurance), maximal isometric plantar flexion (strength) and range of ankle motion (ROAM); these tests were conducted at baseline, week 6 and week 12. The sample for the study was drawn from the Princess Alexandra Hospital in Brisbane, Australia. Participants with venous leg ulceration who met the inclusion criteria were recruited. The participants were screened via duplex scanning and ankle brachial pressure index (ABPI) to ensure they did not have any arterial complications. Participants were excluded if there was evidence of cellulitis. Demographic data were obtained from each participant and details regarding medical history, quality of life and geriatric depression scores were collected at baseline. Both the intervention and control group were required to complete a weekly exercise diary to monitor activity levels between groups. To test for the effect of the intervention over time, a repeated measures analysis of variance was conducted on the major outcome variables. Group (intervention versus control) was the between subject factor and time (baseline, week 6, week 12) was the within subject or repeated measures factor. Due to the small sample size, further tests were conducted to check the assumptions of the statistical test to be used. The results showed that Mauchly.s Test, the Sphericity assumptions of repeated measures for ANOVA were met. Further tests of homogeneity of variance assumptions also confirmed that this assumption was met. Data analysis was conducted using the software package SPSS for Windows Release 17.0. The pilot study proved feasible with all of the intervention (n=6) participants continuing with the resistance program for the 12 week duration and no deleterious effects noted. Clinical significance was observed in the intervention group with a 32% greater change in ulcer size (p= 0.26) than the control group, and a 10% (p = 0.74) greater difference between the numbers healed compared to the control group. Statistical significance was observed for the ejection fraction (p = 0.05), residual volume fraction (p = 0.04) and ROAM (p = 0.01), which all improved significantly in the intervention group over time. These results are encouraging, nevertheless, further investigations seem warranted to examine the effect exercise has on the healing rates of venous leg ulcers, with a multistudy site, larger sample size and longer follow up period.

Monitoring mesenchymal stem cell cultures using image processing and pattern recognition techniques

Stem cells have attracted tremendous interest in recent times due to their promise in providing innovative new treatments for a great range of currently debilitating diseases. This is due to their potential ability to regenerate and repair damaged tissue, and hence restore lost body function, in a manner beyond the body's usual healing process. Bone marrow-derived mesenchymal stem cells or bone marrow stromal cells are one type of adult stem cells that are of particular interest. Since they are derived from a living human adult donor, they do not have the ethical issues associated with the use of human embryonic stem cells. They are also able to be taken from a patient or other donors with relative ease and then grown readily in the laboratory for clinical application. Despite the attractive properties of bone marrow stromal cells, there is presently no quick and easy way to determine the quality of a sample of such cells. Presently, a sample must be grown for weeks and subject to various time-consuming assays, under the direction of an expert cell biologist, to determine whether it will be useful. Hence there is a great need for innovative new ways to assess the quality of cell cultures for research and potential clinical application. The research presented in this thesis investigates the use of computerised image processing and pattern recognition techniques to provide a quicker and simpler method for the quality assessment of bone marrow stromal cell cultures. In particular, aim of this work is to find out whether it is possible, through the use of image processing and pattern recognition techniques, to predict the growth potential of a culture of human bone marrow stromal cells at early stages, before it is readily apparent to a human observer. With the above aim in mind, a computerised system was developed to classify the quality of bone marrow stromal cell cultures based on phase contrast microscopy images. Our system was trained and tested on mixed images of both healthy and unhealthy bone marrow stromal cell samples taken from three different patients. This system, when presented with 44 previously unseen bone marrow stromal cell culture images, outperformed human experts in the ability to correctly classify healthy and unhealthy cultures. The system correctly classified the health status of an image 88% of the time compared to an average of 72% of the time for human experts. Extensive training and testing of the system on a set of 139 normal sized images and 567 smaller image tiles showed an average performance of 86% and 85% correct classifications, respectively. The contributions of this thesis include demonstrating the applicability and potential of computerised image processing and pattern recognition techniques to the task of quality assessment of bone marrow stromal cell cultures. As part of this system, an image normalisation method has been suggested and a new segmentation algorithm has been developed for locating cell regions of irregularly shaped cells in phase contrast images. Importantly, we have validated the efficacy of both the normalisation and segmentation method, by demonstrating that both methods quantitatively improve the classification performance of subsequent pattern recognition algorithms, in discriminating between cell cultures of differing health status. We have shown that the quality of a cell culture of bone marrow stromal cells may be assessed without the need to either segment individual cells or to use time-lapse imaging. Finally, we have proposed a set of features, that when extracted from the cell regions of segmented input images, can be used to train current state of the art pattern recognition systems to predict the quality of bone marrow stromal cell cultures earlier and more consistently than human experts.

The influence of maternal ethnic group and diet on breast milk fatty acid composition

INTRODUCTION: Breast milk fatty acids play a major role in infant development. However, no data have compared the breast milk composition of different ethnic groups living in the same environment. We aimed to (i) investigate breast milk fatty acid composition of three ethnic groups in Singapore and (ii) determine dietary fatty acid patterns in these groups and any association with breast milk fatty acid composition. MATERIALS AND METHODS: This was a prospective study conducted at a tertiary hospital in Singapore. Healthy pregnant women with the intention to breastfeed were recruited. Diet profile was studied using a standard validated 3-day food diary. Breast milk was collected from mothers at 1 to 2 weeks and 6 to 8 weeks postnatally. Agilent gas chromatograph (6870N) equipped with a mass spectrometer (5975) and an automatic liquid sampler (ALS) system with a split mode was used for analysis. RESULTS: Seventy-two breast milk samples were obtained from 52 subjects. Analysis showed that breast milk ETA (Eicosatetraenoic acid) and ETA:EA (Eicosatrienoic acid) ratio were significantly different among the races (P = 0.031 and P = 0.020), with ETA being the highest among Indians and the lowest among Malays. Docosahexaenoic acid was significantly higher among Chinese compared to Indians and Malays. No difference was demonstrated in n3 and n6 levels in the food diet analysis among the 3 ethnic groups. CONCLUSIONS: Differences exist in breast milk fatty acid composition in different ethnic groups in the same region, although no difference was demonstrated in the diet analysis. Factors other than maternal diet may play a role in breast milk fatty acid composition.

Structured Telephone Support or Telemonitoring Programs for Patients with Chronic Heart Failure

Background: Specialised disease management programmes for chronic heart failure (CHF) improve survival, quality of life and reduce healthcare utilisation. The overall efficacy of structured telephone support or telemonitoring as an individual component of a CHF disease management strategy remains inconclusive. Objectives: To review randomised controlled trials (RCTs) of structured telephone support or telemonitoring compared to standard practice for patients with CHF in order to quantify the effects of these interventions over and above usual care for these patients. Search strategy: Databases (the Cochrane Central Register of Controlled Trials (CENTRAL), Database of Abstracts of Reviews of Effects (DARE) and Health Technology Assessment Database (HTA) on The Cochrane Library, MEDLINE, EMBASE, CINAHL, AMED and Science Citation Index Expanded and Conference Citation Index on ISI Web of Knowledge) and various search engines were searched from 2006 to November 2008 to update a previously published non-Cochrane review. Bibliographies of relevant studies and systematic reviews and abstract conference proceedings were handsearched. No language limits were applied. Selection criteria: Only peer reviewed, published RCTs comparing structured telephone support or telemonitoring to usual care of CHF patients were included. Unpublished abstract data was included in sensitivity analyses. The intervention or usual care could not include a home visit or more than the usual (four to six weeks) clinic follow-up. Data collection and analysis: Data were presented as risk ratio (RR) with 95% confidence intervals (CI). Primary outcomes included all-cause mortality, all-cause and CHF-related hospitalisations which were meta-analysed using fixed effects models. Other outcomes included length of stay, quality of life, acceptability and cost and these were described and tabulated. Main results: Twenty-five studies and five published abstracts were included. Of the 25 full peer-reviewed studies meta-analysed, 16 evaluated structured telephone support (5613 participants), 11 evaluated telemonitoring (2710 participants), and two tested both interventions (included in counts). Telemonitoring reduced all-cause mortality (RR 0.66, 95% CI 0.54 to 0.81, P < 0.0001) with structured telephone support demonstrating a non-significant positive effect (RR 0.88, 95% CI 0.76 to 1.01, P = 0.08). Both structured telephone support (RR 0.77, 95% CI 0.68 to 0.87, P < 0.0001) and telemonitoring (RR 0.79, 95% CI 0.67 to 0.94, P = 0.008) reduced CHF-related hospitalisations. For both interventions, several studies improved quality of life, reduced healthcare costs and were acceptable to patients. Improvements in prescribing, patient knowledge and self-care, and New York Heart Association (NYHA) functional class were observed. Authors' conclusions: Structured telephone support and telemonitoring are effective in reducing the risk of all-cause mortality and CHF-related hospitalisations in patients with CHF; they improve quality of life, reduce costs, and evidence-based prescribing.