William L. Olbricht

Building drug delivery into tissue engineering design

The creation of efficient methods for manufacturing biotechnology drugs — many of which influence fundamental but complex cell behaviours, such as proliferation, migration and differentiation — is creating new opportunities for tissue repair. Many agents are potent and multifunctional; that is, they produce different effects within different tissues. Therefore, control of tissue concentration and spatial localization of delivery is essential for safety and effectiveness. Synthetic systems that can control agent delivery are particularly promising as materials for enhancing tissue regeneration. This review discusses the state of the art in controlled-release and microfluidic drug delivery technologies, and outlines their potential applications for tissue engineering.

Dilation and degradation of the brain extracellular matrix enhances penetration of infused polymer nanoparticles.

This study investigates methods of manipulating the brain extracellular matrix (ECM) to enhance the penetration of nanoparticle drug carriers in convection-enhanced delivery (CED). A probe was fabricated with two independent microfluidic channels to infuse, either simultaneously or sequentially, nanoparticles and ECM-modifying agents. Infusions were performed in the striatum of the normal rat brain. Monodisperse polystyrene particles with a diameter of 54 nm were used as a model nanoparticle system. Because the size of these particles is comparable to the effective pore size of the ECM, their transport may be significantly hindered compared with the transport of low molecular weight molecules. To enhance the transport of the infused nanoparticles, we attempted to increase the effective pore size of the ECM by two methods: dilating the extracellular space and degrading selected constituents of the ECM. Two methods of dilating the extracellular space were investigated: co-infusion of nanoparticles and a hyperosmolar solution of mannitol, and pre-infusion of an isotonic buffer solution followed by infusion of nanoparticles. These treatments resulted in an increase in the nanoparticle distribution volume of 51% and 123%, respectively. To degrade hyaluronan, a primary structural component of the brain ECM, a pre-infusion of hyaluronidase (20,000 U/mL) was followed after 30 min by infusion of nanoparticles. This treatment resulted in an increase in the nanoparticle distribution of 64%. Our results suggest that both dilation and enzymatic digestion can be incorporated into CED protocols to enhance nanoparticle penetration.

In vivo two-photon excited fluorescence microscopy reveals cardiac- and respiration-dependent pulsatile blood flow in cortical blood vessels in mice

Subtle alterations in cerebral blood flow can impact the health and function of brain cells and are linked to cognitive decline and dementia. To understand hemodynamics in the three-dimensional vascular network of the cerebral cortex, we applied two-photon excited fluorescence microscopy to measure the motion of red blood cells (RBCs) in individual microvessels throughout the vascular hierarchy in anesthetized mice. To resolve heartbeat- and respiration-dependent flow dynamics, we simultaneously recorded the electrocardiogram and respiratory waveform. We found that centerline RBC speed decreased with decreasing vessel diameter in arterioles, slowed further through the capillary bed, and then increased with increasing vessel diameter in venules. RBC flow was pulsatile in nearly all cortical vessels, including capillaries and venules. Heartbeat-induced speed modulation decreased through the vascular network, while the delay between heartbeat and the time of maximum speed increased. Capillary tube hematocrit was 0.21 and did not vary with centerline RBC speed or topological position. Spatial RBC flow profiles in surface vessels were blunted compared with a parabola and could be measured at vascular junctions. Finally, we observed a transient decrease in RBC speed in surface vessels before inspiration. In conclusion, we developed an approach to study detailed characteristics of RBC flow in the three-dimensional cortical vasculature, including quantification of fluctuations in centerline RBC speed due to cardiac and respiratory rhythms and flow profile measurements. These methods and the quantitative data on basal cerebral hemodynamics open the door to studies of the normal and diseased-state cerebral microcirculation.

Cortical Microhemorrhages Cause Local Inflammation but Do Not Trigger Widespread Dendrite Degeneration

Microhemorrhages are common in the aging brain, and their incidence is correlated with increased risk of neurodegenerative disease. Past work has shown that occlusion of individual cortical microvessels as well as large-scale hemorrhages can lead to degeneration of neurons and increased inflammation. Using two-photon excited fluorescence microscopy in anesthetized mice, we characterized the acute and chronic dynamics of vessel bleeding, tissue compression, blood flow change, neural degeneration, and inflammation following a microhemorrhage caused by rupturing a single penetrating arteriole with tightly-focused femtosecond laser pulses. We quantified the extravasation of red blood cells (RBCs) and blood plasma into the brain and determined that the bleeding was limited by clotting. The vascular bleeding formed a RBC-filled core that compressed the surrounding parenchymal tissue, but this compression was not sufficient to crush nearby brain capillaries, although blood flow speeds in these vessels was reduced by 20%. Imaging of cortical dendrites revealed no degeneration of the large-scale structure of the dendritic arbor up to 14 days after the microhemorrhage. Dendrites close to the RBC core were displaced by extravasating RBCs but began to relax back one day after the lesion. Finally, we observed a rapid inflammatory response characterized by morphology changes in microglia/macrophages up to 200 µm from the microhemorrhage as well as extension of cellular processes into the RBC core. This inflammation persisted over seven days. Taken together, our data suggest that a cortical microhemorrhage does not directly cause significant neural pathology but does trigger a sustained, local inflammatory response.

Flexible microfluidic devices supported by biodegradable insertion scaffolds for convection-enhanced neural drug delivery

Convection enhanced delivery (CED) can improve the spatial distribution of drugs delivered directly to the brain. In CED, drugs are infused locally into tissue through a needle or catheter inserted into brain parenchyma. Transport of the infused material is dominated by convection, which enhances drug penetration into tissue compared with diffusion mediated delivery. We have fabricated and characterized an implantable microfluidic device for chronic convection enhanced delivery protocols. The device consists of a flexible parylene-C microfluidic channel that is supported during its insertion into tissue by a biodegradable poly(DL-lactide-co-glycolide) scaffold. The scaffold is designed to enable tissue penetration and then erode over time, leaving only the flexible channel implanted in the tissue. The device was able to reproducibly inject fluid into neural tissue in acute experiments with final infusate distributions that closely approximate delivery from an ideal point source. This system shows promise as a tool for chronic CED protocols.

Fluid mechanics in the perivascular space

Perivascular space (PVS) within the brain is an important pathway for interstitial fluid (ISF) and solute transport. Fluid flowing in the PVS can affect these transport processes and has significant impacts on physiology. In this paper, we carry out a theoretical analysis to investigate the fluid mechanics in the PVS. With certain assumptions and approximations, we are able to find an analytical solution to the problem. We discuss the physical meanings of the solution and particularly examine the consequences of the induced fluid flow in the context of convection-enhanced delivery (CED). We conclude that peristaltic motions of the blood vessel walls can facilitate fluid and solute transport in the PVS.

A micromechanical flow sensor for microfluidic applications

We fabricated a microfluidic flow meter and measured its response to fluid flow in a microfluidic channel. The flow meter consisted of a micromechanical plate, coupled to a laser deflection system to measure the deflection of the plate during fluid flow. The 100 /spl mu/m square plate was clamped on three sides and elevated 3 /spl mu/m above the bottom surface of the channel. The response of the flow meter was measured for flow rates, ranging from 2.1 to 41.7 /spl mu/L/min. Several fluids, with dynamic viscosities ranging from 0.8 to 4.5/spl times/10/sup -3/ N/m, were flowed through the channels. Flow was established in the microfluidic channel by means of a syringe pump, and the angular deflection of the plate monitored. The response of the plate to flow of a fluid with a viscosity of 4.5/spl times/10/sup -3/ N/m was linear for all flow rates, while the plate responded linearly to flow rates less than 4.2 /spl mu/L/min of solutions with lower dynamic viscosities. The sensitivity of the deflection of the plate to fluid flow was 12.5/spl plusmn/0.2 /spl mu/rad/(/spl mu/L/min), for a fluid with a viscosity of 4.5/spl times/10/sup -3/ N/m. The encapsulated plate provided local flow information along the length of a microfluidic channel.

Simplified Mesofluidic Systems for the Formation of Micron to Millimeter Droplets and the Synthesis of Materials

We present and validate simple mesofluidic devices for producing monodisperse droplets and materials. The significance of this work is a demonstration that simple and complex droplet formulations can be prepared uniformly using off-the-shelf small-diameter tubing, barbed tubing adapters, and needles. With these simple tools, multiple droplet-forming devices and a new particle concentrator were produced and validated. We demonstrate that the droplet-forming devices could produce low-dispersity particles from 25 to 1200 μm and that these results are similar to results from more complicated devices. Through a study of the fluid dynamics and a dimensional analysis of the data, we have correlated droplet size with two dimensionless groups, capillary number and viscosity ratio. The flow-focusing device is more sensitive to both parameters than the T-junction geometry. The modular character of our mesofluidic devices allowed us to rapidly assemble compound devices that use flow-focusing and T-junction devices in series to create complex droplet-in-microcapsule materials. This work demonstrates that flow chemistry does not require complicated tools, and an inexpensive tool-kit can allow anyone with interest to enter the field.

A portable high-intensity focused ultrasound device for noninvasive venous ablation

BACKGROUND Varicose veins and other vascular abnormalities are common clinical entities. Treatment options include vein stripping, sclerotherapy, and endovenous laser treatment, but all involve some degree of invasive intervention. The purpose of this study was to determine ex vivo the effectiveness of a novel hand-held, battery-operated, high-intensity focused ultrasound (HIFU) device for transcutaneous venous ablation. METHODS The ultrasound device is 14 x 9 x 4 cm, weighs 650 g, and is powered by 4 lithium ion battery packs. An ex vivo testing platform consisting of two different models comprised of sequentially layered skin-muscle-vein or skin-fat-vein was developed, and specimens were treated with HIFU. The tissues were then disassembled, imaged, and processed for histology. The luminal cross-sectional area of vein that had been treated with HIFU and nontreated controls were measured, and the values presented as median and interquartile range (IQR). The values were compared using a Wilcoxon rank-sum test, and statistical significance was set at P < .05. RESULTS On gross and histologic examination, veins that had been treated with HIFU showed evidence of coagulation necrosis. The surface of the muscle in direct contact with the vein had a pinpoint area of coagulation, whereas the adjacent fat appeared undisturbed; the skin, fat, and the surface of the muscle in contact with the transducer remained completely unaffected. The cross-sectional area was 3.79 mm(2) (IQR, 3.38-4.22) of the control vein lumen and 0.16 mm(2) (IQR, 0.04-0.39) in those that had been treated with HIFU (P = .0304). CONCLUSION This inexpensive, portable HIFU device has the potential to allow clinicians to easily perform venous ablation in a manner that is entirely noninvasive and without the expense or inconvenience of large, complicated devices. This device represents a significant step forward in the development of new applications for HIFU technology.

Design and characterization of a high-power ultrasound driver with ultralow-output impedance

We describe a pocket-sized ultrasound driver with an ultralow-output impedance amplifier circuit (less than 0.05 ohms) that can transfer more than 99% of the voltage from a power supply to the ultrasound transducer with minimal reflections. The device produces high-power acoustical energy waves while operating at lower voltages than conventional ultrasound driving systems because energy losses owing to mismatched impedance are minimized. The peak performance of the driver is measured experimentally with a PZT-4, 1.54 MHz, piezoelectric ceramic, and modeled using an adjusted Mason model over a range of transducer resonant frequencies. The ultrasound driver can deliver a 100 V(pp) (peak to peak) square-wave signal across 0-8 MHz ultrasound transducers in 5 ms bursts through continuous wave operation, producing acoustic powers exceeding 130 W. Effects of frequency, output impedance of the driver, and input impedance of the transducer on the maximum acoustic output power of piezoelectric transducers are examined. The small size, high power, and efficiency of the ultrasound driver make this technology useful for research, medical, and industrial ultrasonic applications.