Biomedical subjects

Timothy M Wick

Publications and source records attributed to Timothy M Wick.

9 recordsLinked to original sources

PubMed2006

Histamine increases sickle erythrocyte adherence to endothelium.

Complications of sickle cell anaemia include vascular occlusion triggered by the adherence of sickle erythrocytes to endothelium in the postcapillary venules. Adherence can be promoted by inflammatory mediators that induce endothelial cell adhesion molecule expression and arrest flowing erythrocytes. The present study characterised the effect of histamine stimulation on the kinetics of sickle cell adherence to large vessel and microvascular endothelium under physiological flow. Increased sickle cell adherence was observed within minutes of endothelial activation by histamine and reached a maximum value within 30 min. At steady state, sickle cell adherence to histamine-stimulated endothelium was 47 +/- 4 adherent cells/mm(2), 2.6-fold higher than sickle cell adherence to unstimulated endothelial cells. Histamine-induced sickle cell adherence occurred rapidly and transiently. Studies using histamine receptor agonists and antagonists suggest that histamine-induced sickle cell adhesion depends on simultaneous stimulation of the H(2) and H(4) histamine receptors and endothelial P-selectin expression. These data show that histamine release may promote sickle cell adherence and vaso-occlusion. In vivo histamine release should be studied to determine its role in sickle complications and whether blocking of specific histamine receptors may prevent clinical complications or adverse effects from histamine release stimulated by opiate analgesic treatment.

Analysis of Variance↗

PubMed2005

Endothelial cell-smooth muscle cell co-culture in a perfusion bioreactor system.

Vascular endothelial cells (EC) are exposed to a complex biomechanical environment in vivo and are responsible for relaying important messages to the underlying tissue. EC and smooth muscle cells (SMC) communicate to regulate vascular development and function. In this work, a vascular perfusion bioreactor is used to grow tubular constructs seeded with EC and SMC under pulsatile shear stress in long-term co-culture to study the effects of EC on SMC function. SMC seeded into porous poly(glycolic acid) tubular scaffolds are cultured in the bioreactor for 25 days. Constructs are seeded with EC on day 10 or day 23 creating 2-day (short-term) or 15-day (long-term) EC and SMC co-cultures. Long-term EC-SMC co-culture significantly increases cell proliferation and downregulates collagen and proteoglycan deposition compared to short-term co-culture. After 25 days of culture, 15-day co-culture constructs have a more uniform cell distribution across the construct thickness and SMC express a more contractile phenotype compared to 2-day co-culture constructs. These data demonstrate strong interactions between SMC and EC in the bioreactor under physiologically relevant conditions. Thus, the vascular construct perfusion bioreactor is an important tool to investigate cell-cell and cell-extracellular matrix interactions in vascular cell biology and tissue engineering.

Animals↗

PubMed2004

Sickle cell adhesion depends on hemodynamics and endothelial activation.

Venular microvascular circulation in patients with sickle cell anemia exhibits reduced and episodic blood flow. Sickle erythrocyte adhesion to postcapillary venule endothelium is postulated to initiate and propagate episodes of vasoocclusive pain. Hemodynamics likely mediate the adherence of sickle cells to endothelium, controlling delivery of potentially adherent erythrocytes and removal of loosely adherent erythrocytes on the endothelium. This study found a high dependence on shear stress of sickle erythrocyte adhesion to vascular cell adhesion molecule-1 (VCAM-1) on endothelium stimulated by tumor necrosis factor-alpha (TNF-alpha). Shear stress varied from 1.0 dyne/cm 2 (microvascular venular flow), in which VCAM-1 ligand interactions induced by TNF-alpha primarily controlled adherence, to 0.1 dyne/cm 2 (low flow), in which stimulation had little effect on adherence. At shear stresses analogous to in vivo velocities from laser Doppler ultrasound studies (0.8 and 0.6 dyne/cm 2 ), TNF-alpha promoted 1.9- and 2.7-fold increases in adhesion compared with unstimulated (baseline) adherence. These findings suggest a dynamic vasoocclusive process that depends on both receptor expression and shear stress. These results indicate that, in the microvasculature, slightly reduced inflow rate, increased receptor expression, or both may result in large increases in sickle erythrocyte adhesion.

Anemia, Sickle Cell↗

PubMed2003

Inflammatory mediators promote strong sickle cell adherence to endothelium under venular flow conditions.

Adherence of sickle erythrocytes to endothelium in venules is thought to initiate or propagate vaso-occlusive episodes. Because of blood shear forces with normal microvascular flow, adherence in post-capillary venules requires binding via high-affinity receptor-mediated pathways. Microvascular flow in sickle patients is episodic, even in asymptomatic patients, so adherence may also occur at low shear not requiring high-affinity binding. Sickle cell binding to endothelium was quantified under flow or static incubation with unusually large vWF, thrombospondin, alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/fibronectin (FN). Adherence under flow at 0.5 dyne/cm(2) shear stress leads to the greatest number of adherent sickle cells. Adherence under flow at 1.0 dyne/cm(2) leads to the strongest adherence. Static incubation conditions promote weak adherence of low numbers of sickle cells to endothelium. Following attachment at 1.0 dyne/cm(2), adherence strength was 2.5 +/- 0.1 or 2.6 +/- 0.2 dynes/cm(2) for alpha(4)beta(1)/VCAM-1 or alpha(4)beta(1)/FN pathways, a level 50% greater than adherence strength mediated by thrombospondin or ULvWF (1.7 +/- 0.08 or 1.6 +/- 0.07 dynes/cm(2), respectively). Sickle cell adhesion promoted by simultaneous activation of alpha(4)beta(1)/VCAM-1 and alpha(4)beta(1)/FN pathways is the strongest at 6.2 +/- 0.2 dynes/cm(2) and adherent red cells resist detachment shear stresses up to 10 dynes/cm(2). These data demonstrate that sickle cell adhesion to endothelium is regulated both by receptor/ligand affinity and flow conditions. Thus, both microvascular flow conditions and receptor-ligand interactions may regulate sickle cell adherence in vivo.

Anemia, Sickle Cell↗

PubMed2002

Sickle erythrocyte adherence to endothelium at low shear: role of shear stress in propagation of vaso-occlusion.

Under venular flow conditions, sickle cell adherence to endothelium is mediated by cell adhesion molecules and adhesive proteins associated with inflammation, coagulation, and endothelial perturbation. Periodic and reduced blood flow are observed in sickle microcirculation during hematologic steady state, suggesting that blood flow is compromised in sickle microcirculation. We tested the hypothesis that low blood flow enhances adherence by quantifying sickle cell adhesion to endothelium under venular flow (1.0 dyne/cm(2) shear stress) and low flow (0.1 dyne/cm(2) shear stress), with and without addition of adhesion promoting agonists. Under low flow, sickle cell adherence to endothelium increases with contact time in the absence of endothelial activation or adhesive protein addition. In contrast, at venular shear stress, sickle cell adherence only occurs following endothelial activation with TNF-alpha or addition of thrombospondin. Analysis of these data with a mathematical model reveals that at low flow adherence is "transport-controlled," meaning that contact time between sickle cells and endothelium is a more important determinant of adherence than high-affinity receptor-ligand interactions. Low-affinity interactions are sufficient for adhesion at low flow. In contrast, at venular flow (1 dyne/cm(2) shear stress) adherence is "affinity-controlled," meaning that adherence requires induction of specific high-affinity receptor-ligand interactions. These findings demonstrate that in addition to activating factors and adherence proteins, microvascular shear stress is an important determinant of sickle cell adhesion to endothelium. This suggests that in vivo, erythrostasis is an important determinant of adhesion that can act either independently or concurrently with ongoing acute events to induce adhesive interactions and vaso-occlusion.

Anemia, Sickle Cell↗

PubMedDate not supplied

Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering.

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform. Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.

Bioreactors↗

PubMedDate not supplied

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development.

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.

Adaptation, Physiological↗

PubMedDate not supplied

Perfusion bioreactor for small diameter tissue-engineered arteries.

A scaleable perfusion bioreactor has been developed for tissue engineering of small diameter arterial constructs. This modular bioreactor allows for dynamic sequential seeding of smooth muscle and endothelial cells, biomechanical stimulation of cells during culture, and monitoring of tissue growth and maturation. Bovine aortic smooth muscle and endothelial cells were seeded onto porous tubular poly(glycolic acid) nonwoven scaffolds and cultured in the bioreactor under pulsatile flow conditions for up to 25 days. Cell proliferation was more than 3-fold after 4 days, smooth muscle cells expressed differentiated phenotype after 16 days, and collagen and elastin were distributed throughout the construct after 25 days of culture. In bioreactor experiments in which the construct lumen was seeded with endothelial cells by perfusion after 13 days of smooth muscle cell culture, endothelial cell seeding efficiency was 100%, and a confluent monolayer was observed in the lumen within 48 h. These data demonstrate that this perfusion bioreactor supports sequential seeding of constructs with smooth muscle and endothelial cells. Dynamic culture under pulsatile flow leads to cellular expression of differentiated function and extracellular matrix deposition toward the development of tissue-engineered arterial constructs.

Animals↗

PubMedDate not supplied

Effect of low oxygen tension on tissue-engineered cartilage construct development in the concentric cylinder bioreactor.

Cartilage is exposed to low oxygen tension in vivo, suggesting culture in a low-oxygen environment as a strategy to enhance matrix deposition in tissue-engineered cartilage in vitro. To assess the effects of oxygen tension on cartilage matrix accumulation, porous polylactic acid constructs were dynamically seeded in a concentric cylinder bioreactor with bovine chondrocytes and cultured for 3 weeks at either 20 or 5% oxygen tension. Robust chondrocyte proliferation and matrix deposition were achieved. After 22 days in culture, constructs from bioreactors operated at either 20 or 5% oxygen saturation had similar chondrocyte densities and collagen content. During the first 12 days of culture, the matrix glycosaminoglycan (GAG) deposition rate was 19.5 x 10(-9) mg/cell per day at 5% oxygen tension and 65% greater than the matrix GAG deposition rate at 20% oxygen tension. After 22 days of bioreactor culture, constructs at 5% oxygen contained 4.5 +/- 0.3 mg of GAG per construct, nearly double the 2.5 +/- 0.2 mg of GAG per construct at 20% oxygen tension. These data demonstrate that culture in bioreactors at low oxygen tension favors the production and retention of GAG within cartilage matrix without adversely affecting chondrocyte proliferation or collagen deposition. Bioreactor studies such as these can identify conditions that enhance matrix accumulation and construct development for cartilage tissue engineering.

Animals↗