Biomedical subjects

G A Truskey

Publications and source records attributed to G A Truskey.

At least 19 recordsLinked to original sources

PubMed2000

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system.

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.

Animals↗

PubMed2000

Orientation and length of mammalian skeletal myocytes in response to a unidirectional stretch.

Effects of mechanical forces exerted on mammalian skeletal muscle cells during development were studied using an in vitro model to unidirectionally stretch cultured C2C12 cells grown on silastic membrane. Previous models to date have not studied these responses of the mammalian system specifically. The silastic membrane upon which these cells were grown exhibited linear strain behavior over the range of 3.6-14.6% strain, with a Poisson's ratio of approximately 0.5. To mimic murine in utero long bone growth, cell substrates were stretched at an average strain rate of 2.36%/day for 4 days or 1.77%/day for 6 days with an overall membrane strain of 9.5% and 10.6%, respectively. Both control and stretched fibers stained positively for the contractile protein, alpha-actinin, demonstrating muscle fiber development. An effect of stretch on orientation and length of myofibers was observed. At both strain rates, stretched fibers aligned at a smaller angle relative to the direction of stretch and were significantly longer compared to randomly oriented control fibers. There was no effect of duration of stretch on orientation or length, suggesting the cellular responses are independent of strain rate for the range tested. These results demonstrate that, under conditions simulating mammalian long bone growth, cultured myocytes respond to mechanical forces by lengthening and orienting along the direction of stretch.

Animals↗

PubMed2000

Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells.

This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0. 46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.

Biophysical Phenomena↗

PubMed2000

Total internal reflection microscopy and atomic force microscopy (TIRFM-AFM) to study stress transduction mechanisms in endothelial cells.

The cytoskeleton plays a key role in providing strength and structure to the cell. A force balance exists between the cytoskeleton and the extracellular matrix/substratum via the focal contact regions. The purpose of this study is to integrate atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) data to determine the effect of localized force application over the cell surface on the cell's focal contacts size and position. TIRFM gives detailed information on the cell-substrate contact regions and AFM is a tool for elasticity measurements, force application, and topographic surface mapping of the cell. TIRFM data were calibrated by varying the intensity of the evanescent wave to change the interfacial angle at the glass-cell interface. The individual focal contact intensity was found to decrease with increasing interfacial angles from 66 degrees to 80 degrees as the depth of penetration varied from 150 to 66 nm. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire cell using the Bioscope AFM. The nuclear region appears to be stiffer than the cell body. Preliminary results of the nanonewtons force application to the cell surface indicate that the cell-substrate contacts rearrange to offset the force. It is evident that the stress applied to the surface is transmitted to the cell-substrate contact region.

Adaptation, Physiological↗

PubMed1999

Role of endothelial cell-substrate contact area and fibronectin-receptor affinity in cell adhesion to HEMA/EMA copolymers.

The objective of this study was to examine the effect of substrate hydrophobicity on cell-substrate contact area and the affinity between adsorbed fibronectin (Fn) and its receptor. Homo- and copolymer films of hydrophobic ethyl methacrylate (EMA) and hydrophilic hydroxyethyl methacrylate (HEMA) were spun-cast onto glass slides. Bovine aortic endothelial cells (BAEC) were plated for 2 h in serum-free medium onto polymers preadsorbed with Fn. Cells were fixed, labeled, and examined by total internal reflection fluorescence microscopy (TIRFM) to determine the topography of the basal surface as a function of distance from the substrate. Phase contrast microscopy was used to examine the total projected area of adherent cells. The cumulative contact area was greatest on cells attached to surfaces prepared from 0% HEMA and lowest on surfaces with the highest HEMA content. An equilibrium adhesion model used these data together with the critical force for detachment and the Fn density (Burmeister et al., J Biomed Mater Res 1996;30:13-22) to determine the affinity between Fn and its receptor and the bond strength. The affinity and force per bond decreased with increasing HEMA content. These results indicate that differences in the strength of endothelial cell adhesion to polymers are influenced by the conformation of the adsorbed adhesion proteins.

Animals↗

PubMed1999

Relation between non-uniform hemodynamics and sites of altered permeability and lesion growth at the rabbit aorto-celiac junction.

Using the rabbit's aorto-celiac junction as a representative atherosclerotic model, the hemodynamics of a bifurcating blood vessel are numerically simulated and three hemodynamic parameters are compared. The wall shear stress (WSS), the oscillatory shear index (OSI), and the spatial wall shear stress gradient (WSSG) are considered in this study. Locally enhanced wall permeabilities and intimal macrophages are generally considered to be involved in atherogenesis, and here the primary concern is with the hemodynamic influence on these early stages of the disease process. In comparing the segmental averages of the indicator functions and previously published intimal white blood cell densities, only the WSSG shows a statistically significant correlation. All three indicators have selective strengths in determining sites of early lesion growth around the aorto-celiac flow divider. At the proximal end of the flow divider on the lateral side of the orifice, there are elevated values of the OSI as well as WSSG and low WSS values. Regions of elevated wall permeabilities compare with the regions of elevated WSSG along the lateral and distal portions of the flow divider. Largely dependent upon the present input pulse with reverse flow, the OSI indicates relatively high values throughout the flow domain, however, it is important when utilized in conjunction with low WSS regions. This study presents a rationale for further quantitative correlative studies in the rabbit model based on additional histological data sets.

Animals↗

PubMed1999

An equilibrium model of endothelial cell adhesion via integrin-dependent and integrin-independent ligands.

Endothelial cell adhesion can be enhanced by supplementing integrin-mediated adhesion via fibronectin with the high-affinity avidin-biotin system in which biotin is covalently linked to membrane proteins and avidin binds to biotinylated surfaces (Bhat et al. J Biomed Mater Res 1998;41:377-85). An equilibrium model was extended to explain detachment of spreading cells following exposure to flow for this two ligand system. The two different receptor-ligand systems were treated as springs in parallel in which the equilibrium dissociation constant was a function of the separation distance of the cell from the surface. Flow experiments were performed to measure the endothelial cell adhesion strength as a function of the extent of biotinylation of the endothelium. Surfaces contained adsorbed fibronectin, avidin or both ligands. The contact area between the cell membrane and substrate was measured using total internal reflection fluorescence microscopy. Estimates of the unstressed dissociation constant for fibronectin and avidin were determined from data for adhesion strength and contact area of each ligand separately. Using these unstressed equilibrium constants, the model predicted, with reasonable accuracy, the strength of endothelial cell adhesion to surfaces containing fibronectin and avidin. The results indicate that as the extent of biotinylation increases, the avidin-biotin system contributes a larger fraction of the total adhesion strength but the maximum contribution of the avidin-biotin system is less than 50%. The magnitude of the affinity constant and force per bond for the avidin-biotin system are consistent with detachment by extraction of receptors from the cell. The resulting increase in the adhesion strength on surfaces with both avidin-biotin and fibronectin is due to the increase in contact area and the larger number of bonds formed.

Animals↗

PubMed1999

Focal increases in vascular cell adhesion molecule-1 and intimal macrophages at atherosclerosis-susceptible sites in the rabbit aorta after short-term cholesterol feeding.

We tested the hypotheses that vascular cell adhesion molecule-1 (VCAM-1) expression on endothelium at lesion-prone sites in the rabbit aorta correlates with exposure to plasma cholesterol and that macrophage accumulation is associated with endothelial cells expressing VCAM-1. After rabbits were fed 0.25% cholesterol for 2 weeks, VCAM-1 expression was selectively increased at the distal and lateral portions of the major abdominal branches. In the arch and the celiac, superior mesenteric, and renal artery branches, VCAM-1 expression was positively correlated with the plasma cholesterol integrated over the duration of the experiments. After 2 weeks of cholesterol feeding, more macrophages were present around distal and lateral portions of the intercostal arteries and major abdominal branches relative to nonbranch regions. In the arch and around the intercostals and major abdominal branches, macrophage densities were positively correlated with the integrated plasma cholesterol. VCAM-1 and macrophage levels were correlated in lesion-prone regions. In normocholesterolemic rabbits, 23+/-4% (mean+/-SEM) of the macrophages were directly associated with VCAM-1-positive endothelium. After 2 weeks of 0.25% cholesterol feeding, the association increased to 37+/-4% (P<0.015). Associations were highest around the lateral and distal regions of the major abdominal branches. These results suggest that (1) VCAM-1 expression and intimal macrophage densities are influenced by plasma cholesterol and regional factors such as arterial fluid dynamics and (2) VCAM-1 plays a significant role in the localization of macrophages.

Animals↗

PubMed1998

Fibronectin and avidin-biotin as a heterogeneous ligand system for enhanced endothelial cell adhesion.

A preadsorbed layer of "heterogeneous" integrin-dependent and -independent protein was used to enhance initial integrin-mediated endothelial cell attachment and spreading. Glass substrates were treated with fibronectin (Fn) and avidin coupled through adsorbed biotinylated bovine serum albumin (b-BSA). The slides then were seeded with biotinylated BAEC. Control "homogeneous" surfaces were slides adsorbed with either Fn or avidin coupled to b-BSA. The cells were incubated for 0.5 h in serum-containing media and exposed to a range of shear stresses in a laminar flow variable-height flow chamber. The critical shear stress to detach 50% of the seeded cells on the heterogeneous ligand surface was significantly greater than for either of the control homogeneous ligand systems (p < 0.001). Cellular spreading during the initial period of 0-2 h also was higher (p < 0.05) on the heterogeneous ligand-treated surface than on the surface of either of the homogeneous controls. The close contact area of the cell membrane with the substrate 1 h after seeding in serum-containing media was measured using TIRFM. Cells attached onto the heterogeneous ligand-treated surfaces had a significantly (p < 0.01) higher area of close contact with the substrate, which is consistent with a greater degree of attachment and spreading. The results indicate that the combination of integrin-dependent and -independent adhesion systems using heterogeneous ligands further enhances initial endothelial cell attachment and spreading.

Animals↗

PubMed1998

Engineering the tissue which encapsulates subcutaneous implants. II. Plasma-tissue exchange properties.

This study assesses the plasma-tissue exchange characteristics of the capsular tissue that forms around implants and how they are affected by implant porosity. The number of vessels and their permeability to rhodamine were measured by intravascular injection of the fluorophore tracer into Sprague-Dawley rats that hosted for 3-4 months polyvinyl alcohol (PVA) and polytetrafluoroethylene (PTFE) subcutaneous implants. Rats were implanted with four pore sizes of PVA--a nonporous PVA (PVA-skin), and 5, 60, and 700 micron mean pore sizes (PVA-5, PVA-60, and PVA-700, respectively)--and two pore sizes of PTFE: 0.50 (PTFE-0.5) and 5.0 (PTFE-5) mean micron pore sizes. Photodensitometric image analysis was used to quantify the local tracer extravasation and, hence the permeability coefficients of isolated vessels around the implants. The number of functional vessels within 100 microm of the implants highlighted by the lissamine-rhodamine tracer were counted with fluorescence microscopy and with H&E stained sections using brightfield microscopy. The permeability of vessels did not vary substantially with implant pore size but generally were lower than those measured for surrounding subcutis. Pore size, however, had a dramatic effect on the vascular density of tissue-encapsulating implants: the number of microvessels (under 10 microm in radius) within the tissue surrounding the porous implants was higher than the number around nonporous implants. Pore sizes on the order of cellular dimensions incited optimal neovascularization; the vascular density around PVA-60 implants was six times higher (p < .001) and three times higher (p < .001) than those around PVA-0 implants in the fluorescent images and in brightfield, respectively. Moreover, brightfield microscopy showed the number of vessels around PVA-60 implants was almost double those in normal subcutis. The results suggest that optimal vascular density around long-term implants, such as sensors, biofluid cell constructs, and immunoisolated cell systems, may be engineered with pore size.

Animals↗

PubMed1998

Engineering the tissue which encapsulates subcutaneous implants. III. Effective tissue response times.

The results of two previous studies have shown that implant porosity can be used to increase both the measured diffusion coefficients and the vascularity within the tissue encapsulating long-term subcutaneous implants. This study investigates the hypothesis that the analyte concentrations within the tissue surrounding porous implants will respond more quickly to changes in plasma levels than does the densely packed, avascular fibrous capsule surrounding nonporous implants. The average concentration of lissamine-rhodamine was measured in tissue within 100 microm of the following implants at four different times following injection of the tracer: PVA-skin, PVA-5, PVA-60, PVA-700 (polyvinyl alcohol nonporous, 5 microm, 60 microm, and 700 microm mean pore sizes, respectively) and PTFE-0.5 and PTFE-5 (polytetrafluoroethylene 0.5 microm and 5 microm mean pore sizes, respectively). The results were compared to those of unimplanted subcutaneous tissue (SQ). In addition, the data were analyzed with a simple two-compartment model in which a tissue response time constant (taup) was extracted. As in the case of vascular density, the cellular dimension of the PVA-60 pore sizes produced surrounding tissue with the optimum response times to changes in plasma concentrations. The concentrations of rhodamine within the tissue surrounding the PVA-60 implant were the highest at all time points and responded to the change in plasma rhodamine concentration approximately three times more quickly (taup = 764 s) than the fibrous tissue encapsulating the nonporous PVA-skin (taup = 2058 s) and more than twice as quickly as SQ (taup = 1627 s). The overall mass transfer rate between plasma and the tissue surrounding the different implants calculated from the permeability and density of vessels from the previous study correlated very well (r2 = 0.7, p < .02, slope of 0.98) with the reciprocal of the tissue response time constant (taup).

Animals↗

PubMed1998

Using avidin-mediated binding to enhance initial endothelial cell attachment and spreading.

Binding between the protein avidin and the vitamin biotin was used as an extrinsic, high affinity receptor-ligand system to augment the intrinsic integrin-dependent cellular adhesion mechanism. Glass substrates were coupled with avidin receptors through an adsorbed film of biotinylated bovine serum albumin (b-BSA). The avidin-treated slides then were seeded with biotinylated bovine aortic endothelial cells (BAEC). A 3:1 ratio of BSA:b-BSA provided the best results in terms of specific cellular attachment, growth, and spreading. Control surfaces consisted of bare glass or glass with adsorbed BSA. Attachment of unmodified BAEC to glass decreased in the presence of anti-beta 1 integrin antibody. Adhesion of biotinylated BAEC to avidin-treated slides was not affected by anti-beta 1 integrin antibody, consistent with integrin-independent avidin-mediated adhesion. The initial rate of cell spreading was greatest for avidin-biotin-mediated adhesion (80.0 +/- 25.6 microns2/h), followed by integrin-dependent cellular adhesion on plain glass (35.7 +/- 7.7 microns2/h) and, finally, by adhesion on BSA-coated protein surfaces (10.2 +/- 0.3 microns2/h). Biotinylated and unmodified BAEC, cultured for 1 h in serum-containing media, were subjected to laminar flow in a variable-height flow chamber that provided a range of shear stresses from 0.2 to 75 dynes/cm2. The critical shear stress required to detach 50% of the cells in serum-containing media increased from 4.6 +/- 0.8 dynes/cm2 for integrin-dependent adhesion to 12.6 +/- 1.2 dynes/cm2 for avidin-biotin-mediated adhesion. Avidin-mediated attachment for biotinylated BAEC increased initial cellular spreading rates and strength of attachment (i.e., at 1 h) by a factor of two and three, respectively. These results support the hypothesis that integrin-mediated cell attachment and spreading can be enhanced using high affinity integrin-independent binding.

Animals↗

PubMed1998

Association between secondary flow in models of the aorto-celiac junction and subendothelial macrophages in the normal rabbit.

In order to examine the association between arterial fluid dynamics and the distribution of subendothelial macrophages in the normal rabbit aorta, steady and pulsatile particle flow visualization was performed in a geometrically realistic model of the rabbit aorto-celiac junction region. Over a range of aorto-celiac steady flow ratios, particle pathlines along the upstream lateral aortic walls curved to enter the celiac orifice, while two asymmetric regions of reversing spiral secondary flow originated along the downstream lateral portions of the orifice flow divider. These regions increased in size as either the Reynolds number or flow into the celiac artery increased. In pulsatile flow studies, particles along the lateral aortic walls near the celiac orifice began to spiral into the branch during peak systole. During systolic deceleration, the size of this spiral flow region increased as particles reversed direction to enter the celiac orifice. This contrasted with flow patterns directly upstream and downstream of the orifice, which remained unidirectional throughout this period even along the distal lip of the orifice. The highest frequency of subendothelial white blood cells in the normal rabbit aorta was associated with regions where secondary flow patterns occurred, and where the orientation of endothelial cell nuclei deviated from the major direction of aortic flow. Secondary flow patterns may aid the accumulation of monocytes and macrophages about the lateral regions of the celiac artery flow divider by transporting monocytes to the walls, allowing them time to attach to the endothelial cells, or by stimulating the endothelial cells to express leukocyte adhesion molecules. These same regions are associated with increased endothelial permeability to low density lipoprotein and, under hypercholesterolemic conditions, lesion origination.

Animals↗

PubMed1998

Application of total internal reflection fluorescence microscopy to study cell adhesion to biomaterials.

Cell adhesion and function depend upon the formation of adhesive contacts between the cell and substrate. Determination of the cell substrate contact area is necessary in order to understand how biomaterial properties influence cell adhesion. In this review we describe the development and application of total internal reflection fluorescence microscopy (TIRFM) to quantify the separation distance of cells from a biomaterial surface. An approximate theory is presented for the straightforward calculation of separation distances when a fluor is placed in the cell membrane. The validity of this approach is discussed. TIRFM is compared to interference reflection microscopy and related techniques that measure cell/substrate separation distances. This approach is then applied to a number of important problems in cell substrate interactions, including changes in contact area and adhesion strength on biomaterial surfaces, analysis of bond strength, and real-time measurement of cell/substrate separation distances following exposure to flow.

Animals↗

PubMed1998

Effects of recirculating flow on U-937 cell adhesion to human umbilical vein endothelial cells.

We used a sudden-expansion flow chamber to examine U-937 cell adhesion to unactivated and tumor necrosis factor (TNF)-alpha-activated human umbilical vein endothelial cells (HUVEC) in recirculating flow. For both unactivated and TNF-alpha-activated HUVEC, U-937 cells exhibited transient arrests within approximately 150 microm of flow reattachment. Few arrests occurred directly at the reattachment site. U-937 cell rolling was not observed. At all other locations within the recirculation zone, U-937 cells did not exhibit transient arrests or rolling. TNF-alpha activation increased the frequency of U-937 cell arrests near reattachment but did not change the median arrest duration. Numerically simulated cell trajectories failed to predict attachment near the reattachment point. Deviations between experiment and theory may result from the nonspherical shape and deformability of U-937 cells. These results demonstrate that U-937 cell transient arrests occur preferentially in the vicinity of the reattachment point in recirculating flow. Possible mechanisms for adhesion include low shear stress, curved streamlines, fluid velocity components normal to the endothelium, and formation of larger contact areas.

Cell Adhesion↗

PubMed1998

Improving endothelial cell adhesion to vascular graft surfaces: clinical need and strategies.

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to existing therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p < 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm(-2). HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mnm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.

Adsorption↗

PubMed1997

Engineering the tissue which encapsulates subcutaneous implants. I. Diffusion properties.

This report uses normal rat subcutis as a reference point to provide a quantitative analysis of small analyte transport through the tissue which encapsulates implants. Polyvinyl alcohol (PVA) with 60- and 350-micron mean pore size (PVA-60, PVA-350), nonporous PVA (PVA-skin), and stainless-steel cage (SS) specimens were implanted in the subcutis of Sprague-Dawley rats for 4 weeks to elicit a range of capsular wound-healing tissues. Histologic examination showed that the capsular tissue which formed around PVA-skin and SS specimens was densely fibrous and avascular. That forming around PVA-60 and PVA-350 was less densely fibrous and more vascular. The fibrous content of capsular tissue and subcutis was determined from eosin-stained histologic sections. Dual-chamber diffusion measurements of sodium fluorescein (Mw 376 g/mol) through capsular tissue and normal rat subcutis were used to quantitatively compare the effective diffusion coefficients of small analytes on the order of glucose. The two most fibrous capsular tissues exhibited diffusion coefficients that were statistically (p < 0.05) less than that determined for rat subcutis by 50 and 25% for PVA-skin and SS, respectively. The diffusion coefficients of the less dense capsular tissue which formed around the porous implants were not statistically different from subcutis. The experimentally measured diffusion coefficients of the two most fibrous capsular tissues were closely predicted by a simple two-component diffusion model consisting of an aqueous interstitium with an array of impenetrable bodies equal in volume fraction to the fibrous content of the tissue. This model overestimates the diffusion coefficients measured for the least fibrous tissues. Using the diffusion coefficient measured for the PVA-skin capsular tissue, a finite difference model predicts that a 200-microns-thick capsular layer would increase from 5 to 20 min the time required for subcutaneously implanted sensor to detect 95% of the blood analyte concentration. This study suggests that the fibrous capsule forming around a subcutaneously implanted smooth-surface sensor imposes a significant diffusion barrier to small analytes such as glucose, thus increasing the lag time of the sensor by as much as threefold. A corollary observation is that a sensor with a porous surface which allows tissue ingrowth may be more responsive to blood analyte fluctuations as a result of its a more vascular and less fibrous encapsulation tissue.

Animals↗

PubMed1996

Effect of fibronectin amount and conformation on the strength of endothelial cell adhesion to HEMA/EMA copolymers.

The effect of substrate surface hydrophobicity on fibronectin (Fn) adsorption and endothelial cell adhesion strength was studied. Bovine aortic endothelial cells (BAEC) were plated for 2 h with and without preadsorbed Fn on slides coated with homopolymers and copolymers of hydrophilic polyhydroxyethylmethacrylate (polyHEMA) and hydrophobic polyethylmethacrylate (polyEMA). The polarity of the substrate was determined by Wilhelmy plate contact angle. The amount of adsorbed Fn was determined using 125I-labeled Fn. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to detect gross conformational changes of adsorbed Fn on polyHEMA or polyEMA. BAEC were cultured in serum-free medium for 2 h and subjected to a brief exposure of laminar flow in a variable-height flow chamber that provided a range of shear stresses of 15-185 dynes/cm2. The critical shear stress to detach 50% of the cells increased with increasing EMA content to a maximum at 20% HEMA/80% EMA copolymer irrespective of the presence of preadsorbed Fn. However, the critical force increased even though there were similar amounts of Fn adsorbed on all substrates. ATR-FTIR spectroscopy showed only minor changes in beta-sheet structure of Fn adsorbed to polyHEMA and polyEMA. These results show that the force to detach cells did not increase solely with increasing amounts of adsorbed Fn; rather, these results indicate a more complex interplay involving both the amount and conformation of adsorbed Fn.

Animals↗