Biorheology

We have shown that drag-reducing polymers (DRP) reduce microvascular resistance and improve myocardial perfusion during coronary stenosis. We used myocardial contrast echocardiography (MCE) and mathematical modeling to define the DRP microvascular effects. A non-flow-limiting left anterior descending (LAD) stenosis was created in 8 dogs. Intramyocardial blood volume, RBC velocity and flow in the LAD and circumflex (CX) beds were obtained from MCE at baseline, and in hyperemia, stenosis, hyperemia + stenosis, and hyperemia + stenosis + DRP. Microvascular resistances were calculated from a lumped-parameter model. During stenosis + hyperemia, LAD bed microvascular resistance increased (p<0.015), and capillary volume (p<0.002) and red cell velocity (p<0.0004) decreased relative to baseline. With DRP, during stenosis and hyperemia, LAD bed microvascular resistance decreased (p<0.04); there was an increase in capillary volume (p<0.007), RBC velocity (p<0.006), and flow (p<0.05). Decreased model-computed capillary resistance accounted for the reduction in LAD bed resistance after DRP. We conclude that DRP improve flow reserve during coronary stenosis by modulating capillary resistance. Primary modification of the rheological properties of blood to affect capillary resistance is a novel approach for the treatment of acute coronary syndromes.

Several studies have associated elevated plasma homocysteine (Hcy) levels with higher risk of thrombosis. In order to evaluate possible changes in fibrin strength and deformability mediated by Hcy, the effect of the amino acid on plasma fibrin clot was studied, measuring the viscoelastic clots response as a function of Hcy concentration added to plasma (final concentrations: 0, 50, 100, 250 and 500 μM). Storage (G′) and loss (G″) moduli were significantly higher than control at all Hcy concentrations evaluated in a dose-independent way (G′_{50 μM Hcy}=254±10 Pa vs. 178 ± 30 Pa, p=0.012; G″_{50 μM Hcy}=32±1 Pa vs. 24 ± 2 Pa, p=0.012). The tangent of the phase shift angles tan δ obtained from Hcy-clots with respect to control system proved to be unchanged (tan δ_{50 μM Hcy}=0.130±0.007 Pa vs. 0.150 ± 0.020 Pa, NS). Increases observed in G′ and G″ values allowed us to conclude that Hcy action led to the stiffening of the clots formed in a dose independent way. The higher crosslinking of the fibrin network (higher G′) contributed both to this structural behavior and to a higher compartmentalization and viscosity of the fluid phase (higher G″) of the gel. The lower deformability of the clots formed after Hcy addition was also detected through deformation sweep assays. These material's characteristics may lead to pathological behavior, increasing the chances of obstruction in the blood vessels.

The anabolic effect of dynamic mechanical loading on skeletal architecture has been repeatedly demonstrated, but the cellular and molecular events occurring between load and ultimate bone formation remain obscure. The discovery of sclerostin, an antagonist of Wnt/Lrp5 signaling, and the sclerosing bone dysplasias that result from its mutation suggest its pivotal role in modulating bone formation. We examined expression of Sost mRNA across a variety of clonal cell lines spanning the osteogenic phenotype from immature osteoblast to mature osteocyte. No sclerostin expression was detected in immature MC3T3-E1 osteoblasts and, surprisingly, mature MLO-Y4 osteocytes, whereas immature MLO-A5 osteocytic cells expressed very low levels of Sost. Highest expression was observed in mature UMR 106.01 osteoblasts. We examined the influence of bone morphogenetic proteins on Sost expression. Treatment with BMP-2, -4 or -6 was without effect on Sost in mature MLO-Y4 osteocytes but elicited a robust increase in Sost expression in immature MLO-A5 osteocytes. Oscillatory fluid flow applied to mature UMR 106.01 osteoblasts transiently decreased expression of sclerostin at both the mRNA and protein level. Overall, our results indicate that BMP treatment and in vitro mechanical loading demonstrate opposite effects upon sclerostin expression.

Conventional atomic force microscopy is one of the major techniques to evaluate mechanical properties of cells and subcellular components. The use of a cantilever probe for sample manipulation within the vertical plane often makes absolute positioning of the probe, subject to thermal drift, difficult. In addition, the vertical test is unable to observe changes in the sample structure responsible for mechanical behavior detected by the probe. In the present study, an alternative mechanical tester was developed that incorporated a pair of micro-needles to manipulate a sample in a project plane, allowing acquisition of the accurate probe position and entire sample image. Using a vision-based feedback control, a micro-needle driven by a piezo actuator is moved to give user-defined displacements or forces to sample. To show its usefulness and versatility, three types of viscoelastic measurements on actin stress fibers isolated from smooth muscle cells were demonstrated: strain rate-controlled tensile tests, relaxation tests and creep tests. Fluorescence imaging of the stress fibers using Qdots over the course of the measurements, obtained through multiple image detectors, was also carried out. The technique described here is useful for examining the quantitative relationship between mechanical behavior and related structural changes of biomaterials.

Limitations to nutrient transport provide a challenge to the development of 3D tissue-engineered constructs. A heterogeneous distribution of viable cells and functional matrix within the developing tissue is a common consequence. In the present study, a bioreactor was developed to perfuse fluid through cylindrical agarose constructs. The transport and distribution of dextran molecules (FD-4, FD-500, FD-2000) within the agarose was visualized in order to determine the bioreactors effectiveness for transport enhancement. By 24 h, the perfusion bioreactor achieved 529%, 395% and 294% higher concentrations of FD-4, FD-500 and FD-2000, respectively, than those solely due to diffusion. Of particular interest was the effectiveness of the bioreactor to transport molecules to the central region of the constructs. In this respect, the perfusion bioreactor was found to increase transportation of FD-4, FD-500 and FD-2000 by 30%, 291% and 222% over that of diffusion. Articular chondrocytes were cultured and perfused using the bioreactor. The improved molecular transport achieved led to an average 75% and 1340% increase of DNA and sulphated GAG, respectively at 20 days. More significantly was the 106% and 1603% increase of DNA and GAG, respectively, achieved at the central core of the 3D constructs.