Biomedical subjects

W Monty Reichert

Publications and source records attributed to W Monty Reichert.

7 recordsLinked to original sources

PubMed2007

Glucose recovery with bare and hydrogel-coated microdialysis probes: experiment and simulation of temporal effects.

In vitro microdialysis glucose sampling was used to test the transient and steady-state suitability of antifouling hydrogel coatings, composed of 2-hydroxyethyl methacrylate, vinylpyrrolidinone, and poly(ethylene glycol). The in vitro glucose diffusion coefficients of bare microdialysis membranes and hydrogel coatings were determined experimentally to be 1.1 x 10-6 and 3.2 x 10-6 cm2/s, respectively. These values were used to numerically simulate the effect of the hydrogel on glucose transport across the microdialysis membrane using a convection-diffusion transport model. The times for dialysate at the exit of the bare and hydrogel-coated microdialysis probes to reach 95% of steady state were calculated to be 20 and 66 s, respectively. However, the experimental data showed that 95% of steady-state glucose recoveries were reached after 4-5 min. Numerical simulations incorporating the Taylor dispersion in the outlet tubing showed the time difference was caused almost completely by convective transport in the outlet tubing with negligible contribution from analyte profile broadening. These data indicated that the hydrogel coatings imposed 44% reduction in glucose permeability and consequently 26% reduction in the percent recovery. The effect of hydrogel coatings on the time to reach the steady-state recovery was insignificant compared with the time required for convection of glucose in the outlet tubing.

Glucose↗

PubMed2006

In vitro model of glial scarring around neuroelectrodes chronically implanted in the CNS.

A novel in vitro model of glial scarring was developed by adapting a primary cell-based system previously used for studying neuroinflammatory processes in neurodegenerative disease. Midbrains from embryonic day 14 Fischer 344 rats were mechanically dissociated and grown on poly-D-lysine coated 24 well plates to a confluent layer of neurons, astrocytes, and microglia. The culture was injured with either a mechanical scrape or foreign-body placement (segments of 50 microm diameter stainless steel microwire), fixed at time points from 6 h to 10 days, and assessed by immunocytochemistry. Microglia invaded the scraped wound area at early time points and hypertrophied activated astrocytes repopulated the wound after 7 days. The chronic presence of microwire resulted in a glial scar forming at 10 days, with microglia forming an inner layer of cells coating the microwire, while astrocytes surrounded the microglial core with a network of cellular processes containing upregulated GFAP. Vimentin expressing cells and processes were present in the scrape at early times and within the astrocyte processes forming the glial scar. Neurons within the culture did not repopulate the scrape wound and did not respond to the microwire, although they were determined to be electrically active through patch clamp recording. The time course and relative positions of the glia in response to the different injury paradigms correlated well with stereotypical in vivo responses and warrant further work in the development of a functional in vitro test bed.

Animals↗

PubMed2006

Directed cell migration on fibronectin gradients: effect of gradient slope.

The migration of human microvascular endothelial cells (hMEC) was measured on a range of fibronectin gradient slopes. hMEC drift speed increased with increasing gradient slope with no concurrent change in cellular persistence time or random cell speed. The frequency of discrete cellular motion in the gradient direction increased with gradient slope. Morphological polarization of cells on the gradients is also characterized and correlated with cellular drift speed. These experiments present the first demonstration of cellular response to changing haptotactic gradient slope using an in vitro system for the quantitative study of cell migration.

Actins↗

PubMed2005

Protein array method for assessing in vitro biomaterial-induced cytokine expression.

This study demonstrates the feasibility of a cytokine-based in vitro test for biomaterials. The combination of monocyte culture and protein array technology tested in this study permitted the detection of subtle changes in cytokine expression following an exposure to titanium (Ti) particles. However, a broader range of materials and sample configurations must be examined before these promising results can be translated into a reliable and predictive in vitro biomaterials testing protocol. Modified glass slides were robotically printed with eight identical arrays consisting of capture antibodies against four mouse cytokines [IL-6, TNF-alpha, MIP-2, TGF-beta1] and two positive and two negative detection controls. RAW 264.7 mouse monocytes seeded into six-well plates at 10(5)cells/well were treated with either sterilized Ti particles (test biomaterial), or lipopolysaccharide (LPS; positive control), or untreated (negative control). Aliquots (80 microl) of culture medium collected at 1, 6, 24, 48, and 72 h were applied to the protein arrays for simultaneous sandwich fluoroimmunoassay, followed by imaging the fluorescent intensities on a conventional microarray scanner. LPS induced the release of all four cytokines between 1 and 6h treatment periods, whereas Ti induction of cytokines showed a gradual and subtle increase in cytokine expression for >24 h. Among the four cytokines assayed, TNF-alpha and MIP-2 were most prominently expressed, while IL-6 was slightly elevated and TGF-beta1 was undetected above background.

Animals↗

PubMed2005

Mylar and Teflon-AF as cell culture substrates for studying endothelial cell adhesion.

The textured and opaque nature of Dacron and ePTFE has prevented the use of these fabrics in conventional cell culture techniques normally employed to optimize cell attachment and retention. This lack of optimization has led, in part, to the poor performance of endothelialization strategies for improving vascular graft patency. Here we show that thin, transparent films of Mylar and Teflon-AF are viable in vitro cell culture mimics of Dacron and ePTFE vascular graft materials, particularly for the study of protein mediated endothelial cell (EC) attachment, spreading and adhesion. Glass substrates were used as controls. X-ray photoelectron spectroscopy (XPS) and contact angle analysis showed that Mylar and Teflon-AF have surface chemistries that closely match Dacron and ePTFE. (125)I radiolabeling was used to quantify fibronectin (FN) adsorption, and FN and biotinylated-BSA "dual ligand" co-adsorption onto glass, Mylar and Teflon-AF substrates. Native human umbilical vein endothelial cells (HUVEC) and streptavidin-incubated biotinylated-HUVEC (SA-b-HUVEC) spreading was measured using phase contrast microscopy. Cell retention and adhesion was determined using phase contrast microscopy under laminar flow. All surfaces lacking protein pre-treatment, regardless of surface type, showed the lowest degree of cell spreading and retention. Dual ligand treated Mylar films showed significantly greater SA-b-HUVEC spreading up to 2 h, but were similar to HUVEC on FN treated Mylar at longer times; whereas SA-b-HUVEC spreading on dual ligand treated Teflon-AF was never significantly different from HUVEC on FN treated Teflon-AF at any time point. SA-b-HUVEC retention was significantly greater on dual ligand treated Mylar compared to HUVEC on FN treated Mylar over the entire range of shear stresses tested (3.54-28.3 dynes/cm(2)); whereas SA-b-HUVEC retention to dual ligand and HUVEC retention to FN treated Teflon-AF gave similar results at each shear stress, with only the mid-range of stresses showing significant difference in cell retention to Teflon-AF.

Biocompatible Materials↗

PubMed2004

In vitro characterization of microdialysis sampling of macromolecules.

Experiments were performed to characterize the in vitro collection of macromolecules using microdialysis. Fluorescently labeled proteins and dextrans ranging from 3000 to 150 000 were sampled using a 10-mm, 100 000 molecular weight cutoff, polyethersulfone microdialysis probe. Published models describing microdialysis mass transport of small molecules were examined to determine their appropriateness for sampling of macromolecules. Collection efficiencies, reported as relative recoveries, for macromolecules from 3000 to 70 000 ranged from 5 to 44%. Collection efficiencies determined for microdialysis sampling of macromolecules follow the functionality of published models, although experimental mass transport resistances are to some extent smaller than predicted. Implications of the current study for in vivo microdialysis sampling of cytokines and growth factors are discussed.

Dextrans↗

PubMed2004

Measurement of cell migration on surface-bound fibronectin gradients.

A novel technique for the quantitative observation of cell migration along linear gradient substrates functionalized with adhesive proteins is presented. Gradients of the cell adhesion molecule fibronectin are generated by the cross diffusion of functionalizable alkanethiols on gold and characterized by X-ray photoelectron spectroscopy and surface plasmon resonance. Two distinct migration assays are described that characterize the movement of either sparsely populated noncontacting cells or a confluent monolayer of cells into free space. The drift speed of bovine aortic endothelial cells is measured and shown to increase along a fibronectin gradient when compared to a uniform control substrate using both assays. The results of these experiments establish reproducible conditions for studies of cell migration on gradients of surface-bound ligands.

Animals↗