Sequential venous anastomosis design to enhance patency of arterio-venous grafts for hemodialysis

Arterio-venous grafts (AVGs), the second best option as long-term vascular access for hemodialysis, face major issues of stenosis mainly due to development of intimal hyperplasia at the venous anastomosis which is linked to unfavorable hemodynamic conditions. We have investigated computationally the utility of a coupled sequential venous anastomotic design to replace conventional end-to-side (ETS) venous anastomosis, in order to improve the hemodynamic environment and consequently enhance the patency of AVGs. Two complete vascular access models with the conventional and the proposed venous anastomosis configurations were constructed. Three-dimensional, pulsatile blood flow through the models was simulated, and wall shear stress (WSS)-based hemodynamic parameters were calculated and compared between the two models. Simulation results demonstrated that the proposed anastomotic design provides: (i) a more uniform and smooth flow at the ETS anastomosis, without flow impingement and stagnation point on the artery bed and vortex formation in the heel region of the ETS anastomosis; (ii) more uniform distribution of WSS and substantially lower WSS gradients on the venous wall; and (iii) a spare route for the blood flow to the vein, to avoid re-operation in case of stenosis. The distinctive hemodynamic advantages observed in the proposed anastomotic design can enhance the patency of AVGs.     

An automated laboratory‐scale methodology for the generation of sheared mammalian cell culture samples

Continuous disk‐stack centrifugation is typically used for the removal of cells and cellular debris from mammalian cell culture broths at manufacturing‐scale. The use of scale‐down methods to characterise disk‐stack centrifugation performance enables substantial reductions in material requirements and allows a much wider design space to be tested than is currently possible at pilot‐scale. The process of scaling down centrifugation has historically been challenging due to the difficulties in mimicking the Energy Dissipation Rates (EDRs) in typical machines. This paper describes an alternative and easy‐to‐assemble automated capillary‐based methodology to generate levels of EDRs consistent with those found in a continuous disk‐stack centrifuge. Variations in EDR were achieved through changes in capillary internal diameter and the flow rate of operation through the capillary. The EDRs found to match the levels of shear in the feed zone of a pilot‐scale centrifuge using the experimental method developed in this paper (2.4×10⁵ W/Kg) are consistent with those obtained through previously published computational fluid dynamic (CFD) studies (2.0×10⁵ W/Kg). Furthermore, this methodology can be incorporated into existing scale‐down methods to model the process performance of continuous disk‐stack centrifuges. This was demonstrated through the characterisation of culture hold time, culture temperature and EDRs on centrate quality.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

The use of dinoflagellate bioluminescence to characterize cell stimulation in bioreactors

Bioluminescent dinoflagellates are flow-sensitive marine organisms that produce light emission almost instantaneously upon stimulation by fluid shear in a shear stress dose-dependent manner. In the present study we tested the hypothesis that monitoring bioluminescence by suspended dinoflagellates can be used as a tool to characterize cellular response to hydrodynamic forces in agitated bioreactors. Specific studies were performed to determine: (1) impeller configurations with minimum cell activation, (2) correlations of cellular response and an integrated shear factor, and (3) the effect of rapid acceleration in agitation. Results indicated that (1) at a volumetric mass transfer coefficient of 3 x 10(-4) s(-1), marine impeller configurations were less stimulatory than Rushton configurations, (2) bioluminescence response and a modified volumetric integrated shear factor had an excellent correlation, and (3) rapid acceleration in agitation was highly stimulatory, suggesting a profound effect of temporal gradients in shear in increasing cell stimulation. By using bioluminescence stimulation as an indicator of agitation-induced cell stimulation and/or damage in microcarrier cultures, the present study allows for the verification of hypotheses and development of novel mechanisms of cell damage in bioreactors.     

Combined effects of dynamic tissue shear deformation and insulin-like growth factor I on chondrocyte biosynthesis in cartilage explants

Biophysical forces and biochemical factors play crucial roles in the maintenance of the integrity of articular cartilage. In this study, we explored the effect of dynamic tissue shear deformation and insulin-like growth factor I (IGF-I) on matrix synthesis by chondrocytes within native cartilage explants. Dynamic tissue shear in the range of 0.5-6% strain amplitude at 0.1 Hz was applied to cartilage explants cultured in serum-free medium. Dynamic tissue shear above 1.5% strain amplitude significantly stimulated protein and proteoglycan synthesis, by maximum values of 35 and 25%, respectively, over statically held control specimens. In the absence of tissue shear, IGF-I augmented protein and proteoglycan synthesis up to twofold at IGF-I concentrations in the range of 100-300 ng/ml. When tissue shear and IGF-I stimuli were combined, matrix biosynthesis levels were significantly higher than the maximal effect caused by either stimulus alone. However, there was no significant interaction between tissue shear and IGF-I as determined by two-way ANOVA. We then quantified the effect of dynamic tissue shear on the transport of IGF-I into and within cartilage explants. [125I]IGF-I was added to the medium, and the levels of intratissue [125I]IGF-I were directly measured as a function of time over 48 h in the presence and absence of continuous dynamic shear strain. Dynamic shear did not alter the rate of uptake of [125I]IGF-I into the explants, suggesting that convective diffusion of [125I]IGF-I is negligible under the shear strain conditions used. This is in marked contrast to the enhancement of transport reported in response to uniaxial dynamic compression. Taken together, these data suggest that (1) the stimulatory effect of tissue shear is via mechanotransduction pathways and not by facilitated transport of biochemical factors and (2) chondrocytes may possess complementary signal transduction pathways for biophysical and biochemical factors leading to changes in metabolic activity.     

Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner

We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity.     

Transport features, reaction kinetics and receptor biomechanics controlling selectin and integrin mediated cell adhesion

The distinct and overlapping roles of adhesion molecules belonging to the selectin and integrin families control the rate of leukocyte adhesion to stimulated vascular endothelial cells under hydrodynamic shear flow. Crystal structures have appeared for some of these interactions which complement molecular biology experiments, and clarify the molecular mechanism of the receptor-ligand binding interactions. Binding affinity data have also appeared using surface plasmon resonance and single-molecule biophysics experiments. These studies confirm and extend the predictions of previous experiments carried out in parallel-plate flow chambers, and cone and plate viscometers. This review discusses the current state of understanding on how molecular bond formation rates coupled with cellular and hydrodynamic features regulate leukocyte binding to endothelial cells.     

Modulation of the in vitro angiogenic potential of human mesenchymal stromal cells from different tissue sources

Mesenchymal stromal cells (MSCs) have been widely exploited for the treatment of several conditions due to their intrinsic regenerative and immunomodulatory properties. MSC have demonstrated to be particularly relevant for the treatment of ischemic diseases, where MSC-based therapies can stimulate angiogenesis and induce tissue regeneration. Regardless of the condition targeted, recent analyses of MSC-based clinical trials have demonstrated limited benefits indicating a need to improve the efficacy of this cell product. Preconditioning MSC ex vivo through microenvironment modulation was found to improve MSC survival rate and thus prolong their therapeutic effect. This workstudy aims at enhancing the in vitro angiogenic capacity of a potential MSC-based medicinal product by comparing different sources of MSC and culture conditions. MSC from three different sources (bone marrow [BM], adipose tissue [AT], and umbilical cord matrix [UCM]) were cultured with xenogeneic-/serum-free culture medium under static conditions and their angiogenic potential was studied. Results indicated a higher in vitro angiogenic capacity of UCM MSC, compared with cells derived from BM and AT. Physicochemical preconditioning of UCM MSC through a microcarrier-based culture platform and low oxygen concentration (2% O₂, compared with atmospheric air) increased the in vitro angiogenic potential of the cultured cells. Envisaging the clinical manufacturing of an allogeneic, off-the-shelf MSC-based product, preconditioned UCM MSC maintain the angiogenic gene expression profile upon cryopreservation and delivery processes in the conditions of our study. These results are expected to contribute to the development of MSC-based therapies in the context of angiogenesis.