Biomedical subjects

David A Lee

Publications and source records attributed to David A Lee.

31 records · Page 2Linked to original sources

PubMed2004

Passage in monolayer influences the response of chondrocytes to dynamic compression.

This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.

Cartilage, Articular↗

PubMed2003

Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression.

The temporal response of chondrocyte metabolism in agarose constructs subjected to different dynamic compression regimes was investigated. The current study explored the effects of continuous or intermittent compression using various duty cycles of dynamic compressive loading, over a 48 h culture period. For the continuous compression experiments, duty cycles ranged from 5400 to 172,800 and intermittent compression delivered a total of 86,400 cycles. Large numbers of duty cycles significantly stimulated proteoglycan synthesis with maximal levels obtained for constructs subjected to 12h of intermittent compression. The shortest duration of intermittent compression suggested that further cycles are inhibitory for cell proliferation. Nitrite release was independent of the length or type of compressive regime applied. The uncoupled nature of the metabolic response determined in this study suggests that mechanical conditioning regimes may be fine tuned to selectively stimulate key metabolic parameters of relevance to cartilage tissue engineering.

Animals↗

PubMed2003

Long-distance root-to-shoot transport of phytochelatins and cadmium in Arabidopsis.

Phytochelatin synthases (PCS) mediate cellular heavy-metal resistance in plants, fungi, and worms. However, phytochelatins (PCs) are generally considered to function as intracellular heavy-metal detoxification mechanisms, and whether long-distance transport of PCs occurs during heavy-metal detoxification remains unknown. Here, wheat TaPCS1 cDNA expression was either targeted to Arabidopsis roots with the Arabidopsis alcohol dehydrogenase (Adh) promoter (Adh::TaPCS1/cad1-3) or ectopically expressed with the cauliflower mosaic virus 35S promoter (35S::TaPCS1/cad1-3) in the PC-deficient mutant cad1-3. Adh::TaPCS1/cad1-3 and 35S::TaPCS1/cad1-3 complemented the cadmium, mercury, and arsenic sensitivities of the cad1-3 mutant. Northern blot, RT-PCR, and Western blot analyses showed Adh promoter-driven TaPCS1 expression only in roots and thus demonstrated lack of long-distance TaPCS1 mRNA and protein transport in plants. Fluorescence HPLC analyses showed that under Cd2+ stress, no PCs were detectable in cad1-3. However, in Adh::TaPCS1/cad1-3 plants, PCs were detected in roots and in rosette leaves and stems. Inductively coupled plasma atomic emission spectrometer analyses showed that either root-specific or ectopic expression of TaPCS1 significantly enhanced long-distance Cd2+ transport into stems and rosette leaves. Unexpectedly, transgenic expression of TaPCS1 reduced Cd2+ accumulation in roots compared with cad1-3. The reduced Cd2+ accumulation in roots and enhanced root-to-shoot Cd2+ transport in transgenic plants were abrogated by l-buthionine sulfoximine. The presented findings show that (i) transgenic expression of TaPCS1 suppresses the heavy-metal sensitivity of cad1-3, (ii) PCs can be transported from roots to shoots, and (iii) transgenic expression of the TaPCS1 gene increases long-distance root-to-shoot Cd2+ transport and reduces Cd2+ accumulation in roots.

Aminoacyltransferases↗

PubMed2003

ars1, an Arabidopsis mutant exhibiting increased tolerance to arsenate and increased phosphate uptake.

Arsenic is one of the most toxic pollutants at contaminated sites, yet little is known about the mechanisms by which certain plants survive exposure to high arsenic levels. To gain insight into the mechanisms of arsenic tolerance in plants, we developed a genetic screen to isolate Arabidopsis thaliana mutants with altered tolerance to arsenic. We report here on the isolation of a mutant arsenic resisant 1 (ars1) with increased tolerance to arsenate. ars1 germinates and develops under conditions that completely inhibit growth of wild-type plants and shows a semi-dominant arsenic resistance phenotype. ars1 accumulates levels of arsenic similar to that accumulated by wild-type plants, suggesting that ars1 plants have an increased ability to detoxify arsenate. However, ars1 plants produce phytochelatin levels similar to levels produced by the wild type, and the enhanced resistance of ars1 is not abolished by the gamma-glutamylcysteine synthetase inhibitor l-buthionine sulfoxime (BSO). Furthermore, ars1 plants do not show resistance to arsenite or other toxic metals such as cadmium and chromium. However, ars1 plants do show a higher rate of phosphate uptake than that shown by wild-type plants, and wild-type plants grown with an excess of phosphate show increased tolerance to arsenate. Traditional models of arsenate tolerance in plants are based on the suppression of phosphate uptake pathways and consequently on the reduced uptake of arsenate. Our data suggest that arsenate tolerance in ars1 could be due to a new mechanism mediated by increased phosphate uptake in ars1. Models discussing how increased phosphate uptake could contribute to arsenate tolerance are discussed.

Adaptation, Physiological↗

PubMed2003

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques.

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.

Alginates↗

PubMed2003

Influence of external uniaxial cyclic strain on oriented fibroblast-seeded collagen gels.

This study investigates the influence of cyclic tensile strain, applied to fully contracted fibroblast-seeded collagen constructs. The constructs were preloaded to either 2 or 10 mN. The preloaded constructs were subsequently subjected to a further 10% cyclic strain (0-10%) at 1 Hz, using a triangular waveform, or were cultured in the preloaded state. In all cases cellular viability was maintained during the conditioning period. Cell proliferation was enhanced by the application of cyclic strain within constructs preloaded to both 2 and 10 mN. Collagen synthesis was enhanced by cyclic strain within constructs preloaded at 2 mN only. The profile of matrix metalloproteinase (MMP) expression, determined by zymography, was broadly similar in constructs preloaded at 2 mN with or without the application of cyclic strain. By contrast, constructs preloaded at 10 mN and subjected to cyclic strain expressed enhanced levels of staining for latent MMP-1, latent MMP-9, and both latent and active MMP-2, when compared with the other conditioning regimens. The structural stiffness of constructs preloaded at 2 mN and subjected to cyclic strain was enhanced compared with control specimens, reflecting the increase in collagen synthesis. By contrast, the initial failure loads for cyclically strained constructs preloaded at 10 mN were reduced, potentially because of enhanced catabolic activity.

Collagen↗

PubMed2003

Live cell imaging using confocal microscopy induces intracellular calcium transients and cell death.

Isolated chondrocytes stained with fluo 4-AM and visualized using standard confocal microscopy techniques exhibited Ca2- transients and oscillations. Decreasing the power of the laser light decreased the percent-age of cells exhibiting these Ca2+ signals. Treatment with the antioxidant ascorbate reduced the Ca2+ response, suggesting that it was mediated by light-induced release of reactive oxygen species (ROS). Cell viability 24 h after the 1-h confocal imaging period was approximately 90% for cells that were neither fluorescently stained nor subjected to laser excitation. By contrast, fluorescently stained cells imaged for 1 h exhibited greatly reduced viability. Treatment with ascorbate reduced the level of cell death, suggesting that the effect was mediated by release of exogenous ROS associated with the interaction of light and the fluorochrome. Ca2+ oscillations were not always associated with cell death, suggesting that separate light-sensitive pathways mediate the two processes. Light-activated Ca2+ signaling may trigger alterations in numerous cell processes and thereby represent an important and hitherto overlooked artifact in fluorescent microscopy of viable cells.

Aniline Compounds↗

PubMed2003

Mechanical conditioning influences the metabolic response of cell-seeded constructs.

Injuries and degenerative diseases to load-bearing soft tissues are extremely common in hospital clinics and involve all ages of the population. Tissue engineering provides a major focus for developments within this area due to the relative failure of graft and prosthetic-based strategies. On implantation, such systems will be subjected to normal physiological forces. It is important, therefore, to understand the effects of mechanical conditioning of cells within repair systems to predict their response and ultimate success in vivo. In the current study two well-characterised model systems, involving dermal fibroblasts seeded onto silicone membranes and chondrocytes seeded in agarose constructs, have been used to study the effects of mechanical loading on cellular activity. The data presented demonstrate that both the tensile conditioning of fibroblasts in monolayer and the compressive loading of chondrocytes in three-dimensional agarose constructs produced distinct responses. In particular, the nature of the strain field associated with the tensile system is important in determining the ultimate cell response. Equally within the compressive system the influence of dynamic frequency was shown to be important. The other critical factors in determining response to both types of conditioning are associated with the age of the cell and its original location within the tissue.

Agar↗

PubMed2002

Automated perimetry: a report by the American Academy of Ophthalmology.

OBJECTIVE: The purpose of this document is to summarize and evaluate the effectiveness of new automated perimetry tests and algorithms in diagnosing glaucoma and detecting disease progression. METHODS: A literature search on automated perimetry retrieved over 300 citations from 1994 to 2001, of which 71 were selected as relevant to this assessment. The quality of the evidence obtained from these studies was assessed by the methodologist. RESULTS: The four automated perimetry techniques described in this assessment are short wavelength automated perimetry (SWAP), frequency doubling technology perimetry (FDT), high-pass resolution perimetry (HPRP), and motion automated perimetry (MAP). The algorithms described are Swedish interactive threshold algorithm (SITA) and SITA fast. With the exception of SWAP, these techniques and algorithms reduce testing time and inconsistent patient performance when compared with conventional full threshold testing. CONCLUSIONS: Short wavelength automated perimetry detected visual field loss earlier than standard threshold automated perimetry, with a sensitivity and specificity of about 88% and 92% respectively. However, it is a lengthy, demanding test, is sensitive to media opacities, and has a greater magnitude of long-term fluctuation compared with standard threshold automated perimetry, which make it difficult to assess disease progression accurately. When compared to standard threshold automated perimetry, FDT perimetry showed sensitivity and specificity greater than 97% for detecting moderate and advanced glaucoma, and sensitivity of 85% and specificity of 90% for early glaucoma. As FDT perimetry has a short testing time and is resistant to blur and pupil size, it may be a useful screening tool. In a longitudinal study, high-pass resolution perimetry was more effective than standard threshold automated perimetry in monitoring progressive glaucomatous loss, detecting progression at a median of 12 months earlier in 54% of patients studied. Motion automated perimetry demonstrated usefulness in detecting early glaucomatous visual loss in a longitudinal study. Studies on SITA demonstrated greater sensitivity and reproducibility and less intertest variability when compared to standard full threshold testing and a 50% reduction in testing times. A study comparing standard full threshold, SITA, and SITA fast found a sensitivity of 95% for the first two techniques and 93% for SITA fast. Long-term follow-up studies are needed to assess the ability of these techniques to detect progression of glaucoma over time.

Algorithms↗

PubMed2002

Mycotic aneurysm of the descending thoracic aorta caused by Pseudomonas aeruginosa in a solid organ transplant recipient: case report and review.

BACKGROUND: Pseudomonas aeruginosa is a rare cause of aortic mycotic aneurysms. Optimal treatment, including reconstructive graft material and appropriate length of antibiotic therapy, is being debated. METHODS: We describe a 26-year-old kidney-pancreas recipient who developed an aneurysm of the descending thoracic aorta caused by P. aeruginosa. RESULTS: After surgical debridement and cryopreserved allograft reconstruction, parenteral antibiotics were continued for 12 months, at which time the patient was converted to oral antibiotic therapy. Within 6 months, he redeveloped a thoracic aortic aneurysm, necessitating reoperation and lifelong parenteral antibiotic therapy. CONCLUSION: Herein we review and discuss the relevant literature concerning surgical and antibiotic treatment of mycotic thoracic aneurysms.

Adult↗

PubMedDate not supplied

Cellular utilization determines viability and matrix distribution profiles in chondrocyte-seeded alginate constructs.

The long-term success of any cellular construct used for cartilage tissue engineering is dependent on the maintenance of cell viability throughout the construct thickness. Furthermore, the cells must continue to be metabolically active in order to synthesize a mechanically functional extracellular matrix (ECM). In the present study, a live-dead staining technique and systematic profiling procedure enabled the spatial and temporal distribution of chondrocyte viability to be characterized within 4-mm-thick alginate scaffolds. ECM distribution after 14 days of culture is described both biochemically and histologically and the mechanical functionality of the constructs was assessed by an unconfined compression test. Parameters investigated included alginate permeability, cell-seeding density, and volume of culture medium. Nonhomogeneity of cell and matrix distribution was evident, with greater densities of both parameters in the periphery of the constructs. The culture time preceding central viability loss was inversely related to cell density but relatively independent of scaffold density. However, homogeneity could be attained with increasing medium volume, as evidenced with cell and matrix distribution for cultures in 6.4 mL of medium per 10(6) cells. Moreover, the mechanical properties of the construct were enhanced by culture in increasing volumes of medium. This work indicates that cellular utilization determines the nonhomogeneous nature of cartilage formation in three-dimensional constructs and presents a guide to nonlimiting medium volumes for static culture conditions.

Adaptation, Physiological↗

PubMedDate not supplied

Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs.

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.

Adolescent↗

PubMedDate not supplied

Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells.

This study tests the hypothesis that dynamic compressive strain selectively enhances chondrogenic differentiation by human mesenchymal stem cells (MSCs). Primary MSCs were isolated and expended in monolayer culture. The cells were seeded in alginate constructs or in pellet culture. The time course of chondrogenic differentiation was assessed by real-time QPCR of mRNA expression analysis for cartilage specific markers. Collagen types II and X mRNA, not present in undifferentiated MSCs, were detectable by 2-4 days of chondrogenic induction and continued to rise significantly throughout the culture period of 10 days (p < 0.001). Basal levels of gene expression for Sox-9 and aggrecan were evident in undifferentiated MSCs, although chondrogenic induction for a period of 8 days resulted in an increased trend in the gene expression levels. The alginate system was also used in mechanical conditioning studies. Dynamic compression was applied, in an intermittent regimen, at a strain amplitude of 15% and frequency of 1 Hz in the presence and absence of 10 ng/ml TGFbeta3, for a period of 8 days. Results indicated significant changes in the levels of mRNA expression for the chondrogenic markers. For example, by day 8, the application of the strain regimen alone caused an up-regulation in all the chondrogenic markers compared to the control samples (no TGFbeta, no compression). However, the combined effects of strain and TGFbeta on these markers were more complex than purely additive.

Alginates↗