Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism.

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.

Real-time observation of leukocyte-endothelium interactions in tissue-engineered blood vessel.

Human cell-based 3D tissue constructs play an increasing role in disease modeling and drug screening. Inflammation, atherosclerosis, and many autoimmune disorders involve the interactions between immune cells and blood vessels. However, it has been difficult to image and model these interactions under realistic conditions. In this study, we fabricated a perfusion and imaging chamber to allow the real-time visualization of leukocyte perfusion, adhesion, and migration inside a tissue-engineered blood vessel (TEBV). We monitored the elevated monocyte adhesion to the TEBV wall and transendothelial migration (TEM) as the TEBV endothelium was activated by the inflammatory cytokine TNF-α. We demonstrated that treatment with anti-TNF-α or an NF-kB signaling pathway inhibitor would attenuate the endothelium activation and reduce the number of leukocyte adhesion (>74%) and TEM events (>87%) close to the control. As the first demonstration of real-time imaging of dynamic cellular events within a TEBV, this work paves the way for drug screening and disease modeling in TEBV-associated microphysiological systems.

Normal and shear stresses influence the spatial distribution of intracellular adhesion molecule-1 expression in human umbilical vein endothelial cells exposed to sudden expansion flow.

Patterns in cell adhesion molecule expression by endothelial cells may play a role in atherogenesis. Previous studies have shown dependence of intracellular adhesion molecule-1 (ICAM-1) expression in human umbilical vein endothelial cells (HUVEC) on shear stress and have indirectly linked ICAM-1 expression to spatial gradients in shear stress. The spatial distribution of ICAM-1 in HUVEC pre-exposed to flow for 8h was determined using fluorescence microscopy and a sudden expansion flow chamber with a 2.66 expansion ratio to simulate gradients in wall shear stress found near arterial branches in vivo. When ICAM-1 expression in the disturbed flow region was compared to theoretical stress distributions obtained from a computational model of sudden expansion flow, a modest trend (R2 = 0.327, p < 0.01)was observed between ICAM-1 and shear stress but the correlation between ICAM-1 and shear stress gradient was insignificant. In contrast, a moderately strong trend (R2 = 0.873, p < 0.01) was evident between ICAM-1 expression and the component of normal stress induced by the expansion. Thus, in this in vitro model, normal stress arising from sudden expansion flow modulates the effect of shear stress on ICAM-1 expression.

Human Vascular Microphysiological System for in vitro Drug Screening.

In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 µM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 µM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

The distribution of intimal white blood cells in the normal rabbit aorta.

Macrophages play an important role in atherogenesis and have been reported within the intima at lesion-prone sites in normocholesterolemic animals as well as infants and children. The objective of this study was to determine the spatial distribution of intimal white blood cells (WBC) in the normal rabbit aorta and the association of intimal WBC with replicating endothelial cells and sites of increased 125I-LDL permeability. Intimal WBC and macrophages were identified en face on whole aortic tissue and on Häutchen preparations based on their morphology, ingestion of exogenous horseradish peroxidase, non-specific esterase activity, and labeling with a monoclonal antibody for rabbit macrophages (RAM11). WBC were primarily located in the lesion-prone flow divider regions of the large abdominal branch arteries. Using [3H]thymidine autoradiography to determine cell proliferation, 4.4% of the WBC and 0.12% of the endothelial cells were labeled on the Häutchen preparations. The distribution of replicating endothelial cells was not localized to the arterial orifices and was not correlated with the distribution of intimal WBC. Intimal WBC were, however, spatially correlated with the distribution of 125I-LDL permeable sites about the celiac artery orifice and were directly associated with 31% of the LDL permeable spots. Moreover, mitotic endothelial cells accounted for only 8% of the total number of LDL permeable sites. The presence of intimal WBC at lesion-prone sites in the normocholesterolemic rabbit suggests that these cells may be important in the initiation of atherosclerotic lesions.

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.

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.

Relationship between 3T3 cell spreading and the strength of adhesion on glass and silane surfaces.

Cell detachment by laminar shear stresses was used to characterize cellular interactions with hydrophilic glass and hydrophobic silane. In this study, we examined whether smaller, rounder cells were preferentially detached by laminar flow, and whether cell detachment occurred by dissociation of adhesion proteins and their membrane receptors or rupture of the membrane. Shear-induced detachment from glass and silane were similar after 0.5 h static attachment to the surfaces, even though 3T3 cells had a greater projected area on silane. No particular cell size was preferentially detached by fluid shear stresses. After 2 h attachment and spreading, 3T3 cells were more easily detached from the silane surface even though the cells were more spread than on glass. On glass, smaller cells were preferentially detached below 30 dyne/cm2, increasing the mean projected area of the population. Above 30 dyne/cm2, larger cells also detached from the surface. Cell detachment from the silane surfaces did not show any size preference. The strength of adhesion and projected areas on both surfaces increased significantly when the surfaces were preincubated with fibronectin. Simple geometric models of spreading cells were used to estimate the forces exerted on cells. The hydrodynamic forces exerted on spreading cells were similar, but the bond density needed to resist detachment declined as the projected area increased. Analysis of Dil-C18(3) membrane fragments indicated that cell detachment by membrane rupture was a significant mechanism of cell detachment from glass for shear stresses above 40 dyne/cm2, but was unimportant for cell detachment from the silane surfaces. The results indicate that differences in the strength of 3T3 cell adhesion were probably due to differences in bond strength and the numbers of receptor-ligand bonds formed on the two surfaces and, on glass, cell detachment due to membrane failure at higher shear stresses.

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.

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.

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.

Hemodynamic parameters and early intimal thickening in branching blood vessels.

Intimal thickening due to atherosclerotic lesions or intimal hyperplasia in medium to large blood vessels is a major contributor to heart disease, the leading cause of death in the Western World. Balloon angioplasty with stenting, bypass surgery, and endarterectomy (with or without patch reconstruction) are some of the techniques currently applied to occluded blood vessels. On the basis of the preponderance of clinical evidence that disturbed flow patterns play a key role in the onset and progression of atherosclerosis and intimal hyperplasia, it is of interest to analyze suitable hemodynamic wall parameters that indicate susceptible sites of intimal thickening and/or favorable conditions for thrombi formation. These parameters, based on the wall shear stress, wall pressure, or particle deposition, are applied to interpret experimental/clinical observations of intimal thickening. Utilizing the parameters as "indicator" functions, internal branching blood vessel geometries are analyzed and possibly altered for different purposes: early detection of possibly highly stenosed vessel segments, prediction of future disease progression, and vessel redesign to potentially improve long-term patency rates. At the present time, the focus is on the identification of susceptible sites in branching blood vessels and their subsequent redesign, employing hemodynamic wall parameters. Specifically, the time-averaged wall shear stress (WSS), its spatial gradient (WSSG), the oscillatory shear index (OSI), and the wall shear stress angle gradient (WSSAG) are compared with experimental data for an aortoceliac junction. Then, the OSI, wall particle density (WPD), and WSSAG are segmentally averaged for different carotid artery bifurcations and compared with clinical data of intimal thickening. The third branching blood vessel under consideration is the graft-to-vein anastomosis of a vascular access graft. Suggested redesigns reduce several hemodynamic parameters (i.e., the WSSG, WSSAG, and normal pressure gradient [NPG]), thereby reducing the likelihood of restenosis, especially near the critical toe region.

Quantitation of cell area on glass and fibronectin-coated surfaces by digital image analysis.

By using digital image processing and analysis, two procedures were developed to rapidly measure the projected area of a field of adherent 3T3 fibroblasts without staining of cell borders. The cell area of newly attached and rounded cells with well-resolved borders was obtained by a gray value thresholding procedure. For cells that had undergone an appreciable degree of spreading, cell boundaries were less distinct and a nonlinear spatial Sobel filter was used, followed by thresholding. For both procedures, linear relations were observed between cell areas obtained from image analysis and cell areas obtained by tracing. The areas of a population of traced cells were not statistically different from the area distribution obtained by using the standard curves for the processed images. Uncertainty in the estimated mean area depended only upon the number of cells examined. Approximate numbers of cells required to obtain estimates of the mean are calculated. As an application of these procedures, cell areas were measured for 3T3 cells attached to glass and fibronectin-coated surfaces and were found to be significantly larger for cells spreading on fibronectin-coated glass than on glass alone. Increased cell area during spreading on fibronectin-coated surfaces was proportional to increased cell adhesivity after exposure to a shear stress of 58 dyn/cm2.

Short-term cell/substrate contact dynamics of subconfluent endothelial cells following exposure to laminar flow.

The manner in which fluid stresses are transmitted from the apical to the basal surface of the endothelium will influence the dynamics of cell/substrate contacts. Such dynamics could be important in the design of synthetic vascular grafts to promote endothelial cell adhesion. To examine whether the initial response of cell/substrate contact sites to flow depends on the magnitude of the applied shear stress, subconfluent monolayers of endothelial cells were exposed to flow at 10, 20, and 30 dyn cm-2 wall shear stresses for 20 min. Cell/substrate contact sites were visualized with total internal reflection fluorescence microscopy. Flow induced a rapid fluctuation in the membrane topography, which was reflected in dynamic changes in cell/substrate contacts. Exposure to flow caused marked changes in contact area. Contact movement occurred normal and parallel to the direction of flow. Contact sites demonstrated significant variability in contact area at 30 dyn cm-2 during the experiment but no significant movement of the contact sites in flow direction after 20 min of flow. Mean square displacements of the contact center of mass were described in terms of a directed diffusion model. Prior to onset of flow, contact movement was random. Flow induced a significant convective component to contact movement for 300-600 s, followed by reestablishment of diffusive growth and movement of contacts. These results suggest that fluid stresses are rapidly transmitted from the apical to the basal surface of the cell via the cytoskeleton.

Altered distribution of mitochondria and actin fibers in 3T3 cells cultured on microcarriers.

The mitochondria and actin fibers of 3T3 fibroblasts cultured on microcarriers in spinner flasks were visualized using fluorescent stains. In contrast to cells grown on planar surfaces under static or steady laminar flow conditions, cells exposed to higher levels of turbulent agitation do not form actin stress fibers. Greater agitation also leads to a more diffuse appearance of the mitochondria and a wider distribution of them throughout the cytoplasm. This response may indicate damaged mitochondria, as similar results have been reported for chemical toxins.

Imaging of cell/substrate contacts on polymers by total internal reflection fluorescence microscopy.

A simplified model of total internal reflection fluorescence (TIRF) emission of fluorescently labeled cell membranes [Reichert, W. M.; Truskey, G. A. J. Cell Sci. 1990, 96, 219-230] was used to determine the topography of the cell membrane in apposition to a polymer-coated surface. The homopolymer substrates were spun cast films of hydrophilic poly(hydroxyethyl methacrylate) (polyHEMA) or hydrophobic poly(ethyl methacrylate) (polyEMA). Bovine aortic endothelial cells (BAEC) on preadsorbed fibronectin polymer substrates were either plated for 24 h, fixed, labeled, and examined by TIRF microscopy (TIRFM) and phase-contrast microscopy or plated for 2 h and tested for their adhesion strength in a parallel-plate flow chamber. BAEC attached to polyHEMA showed no evidence of focal contact formation. However, BAEC attached to polyEMA were well spread and showed an array of focal contacts. TIRFM data were transformed to construct a detailed topographical map of relative cell/substrate separation distances. Virtually all of the BAEC plated to polyHEMA were sheared from the surface when subjected to a 50 dyn/cm2 burst of laminar flow, whereas only 10% of the BAEC were sheared from the polyEMA surface. These data suggest that the polyHEMA and polyEMA surface properties (e.g., hydrophobicity) correlate with the presence of BAEC focal contacts and the BAEC attachment strength.

Hemodynamics simulation and identification of susceptible sites of atherosclerotic lesion formation in a model abdominal aorta.

Employing the rabbit's abdominal aorta as a suitable atherosclerotic model, transient three-dimensional blood flow simulations and monocyte deposition patterns were used to evaluate the following hypotheses: (i) simulation of monocyte transport through a model of the rabbit abdominal aorta yields cell deposition patterns similar to those seen in vivo, and (ii) those deposition patterns are correlated with hemodynamic wall parameters related to atherosclerosis. The deposition pattern traces a helical shape down the aorta with local elevation in monocyte adhesion around vessel branches. The cell deposition pattern was altered by an exercise waveform with fewer cells attaching in the upper abdominal aorta but more attaching around the renal orifices. Monocyte deposition was correlated with the wall shear stress gradient and the wall shear stress angle gradient. The wall stress gradient, the wall shear stress angle gradient and the normalized monocyte deposition fraction were correlated with the distribution of monocytes along the abdominal aorta and monocyte deposition is correlated with the measured distribution of monocytes around the major abdominal branches in the cholesterol-fed rabbit. These results suggest that the transport and deposition pattern of monocytes to arterial endothelium plays a significant role in the localization of lesions.

Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy.

This study evaluated the hypothesis that, due to functional and structural differences, the apparent elastic modulus and viscous behavior of cardiac and skeletal muscle and vascular endothelium would differ. To accurately determine the elastic modulus, the contribution of probe velocity, indentation depth, and the assumed shape of the probe were examined. Hysteresis was observed at high indentation velocities arising from viscous effects. Irreversible deformation was not observed for endothelial cells and hysteresis was negligible below 1 microm/s. For skeletal muscle and cardiac muscle cells, hysteresis was negligible below 0.25 microm/s. Viscous dissipation for endothelial and cardiac muscle cells was higher than for skeletal muscle cells. The calculated elastic modulus was most sensitive to the assumed probe geometry for the first 60 nm of indentation for the three cell types. Modeling the probe as a blunt cone-spherical cap resulted in variation in elastic modulus with indentation depth that was less than that calculated by treating the probe as a conical tip. Substrate contributions were negligible since the elastic modulus reached a steady value for indentations above 60 nm and the probe never indented more than 10% of the cell thickness. Cardiac cells were the stiffest (100.3+/-10.7 kPa), the skeletal muscle cells were intermediate (24.7+/-3.5 kPa), and the endothelial cells were the softest with a range of elastic moduli (1.4+/-0.1 to 6.8+/-0.4 kPa) depending on the location of the cell surface tested. Cardiac and skeletal muscle exhibited nonlinear elastic behavior. These passive mechanical properties are generally consistent with the function of these different cell types.

Effects of chronic exposure to simulated microgravity on skeletal muscle cell proliferation and differentiation.

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.

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.