Dorian Liepmann

Regulation of vascular smooth muscle cells by micropatterning.

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.

Clinical microneedle injection of methyl nicotinate: stratum corneum penetration.

Background/purpose: In recent years, microneedles were proposed as a method to painlessly deliver drugs past the stratum corneum. Microneedles have been fabricated in several designs, but limited studies have tested microneedle injections in humans. In this work, we compare microneedle injections with topical application (TA) to investigate if microneedles enhance in vivo drug delivery past the stratum corneum.

Arrays of hollow out-of-plane microneedles for drug delivery

Drug delivery based on MEMS technology requires an invasive interface such as microneedles, which connects the microsystem with the biological environment. Two-dimensional arrays of rigid hollow microneedles have been fabricated from single-crystal silicon using a combination of deep reactive ion etching and isotropic etching techniques. The fabricated needles are typically 200 /spl mu/m long with a wide base and a channel diameter of 40 /spl mu/m. The fabrication process allows creating either blunt needles or needles with sharp tips. Their shape and size make these needles extremely suitable for minimally invasive painless epidermal drug delivery. MEMS technology allows for batch fabrication and integration with complex microsystems. Fluid has been successfully injected 100 /spl mu/m deep into sample tissue through arrays of microneedles. Needle breakage did not occur during this procedure. Experiments have shown that the modified Bernoulli equation is a good model for liquid flowing through the narrow microneedle lumen.

Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel

A basic step in many biological assays is separating and isolating different types of cells from raw samples. To better meet these requirements in microfluidic devices for miniature biomedical analytical systems, an alternative method for separating cells has been devised by mimicking the physiological process of leukocyte recruitment to blood vessel walls: adhesive cell rolling and transient tethering. Reproducing these interactions for cells on surfaces of microstructured fluidic channels can serve to capture and concentrate cells and even to fractionate different cell types from a continuously flowing sample. To demonstrate this principle, two designs for microstructured fluidic channels were fabricated: an array of Square pillars and another with slender, Offset pillars. These structures were coated with E-selectin IgG chimera and the interactions of HL-60 and U-937 cells with these structures were characterized. With inflow of fluidic cell suspensions, the structures were able to efficiently capture and arrest cells directly from the rapid free stream flow. After capture, cells transit through the channel in three phases: cell rolling, cell tethering, and transient re-suspension in free stream flow before re-capture. Under these interactions, captured cells were enriched several hundred-fold from the original concentration. Additionally, among collected cells, the difference in flow-driven, adhesion-mediated cell transit in the Square design suggested that the two cell types could at least be partially fractionated.

Microfabricated Polysilicon Microneedles for Minimally Invasive Biomedical Devices

A two-wafer polysilicon micromolding process has been developed for the fabrication of hollow tubes useful for microfluidic applications. These small tubes can be fabricated with a pointed end, resulting in a micro hypodermic injection needle. Microneedles are desired because they reduce both insertion pain and tissue damage in the patient. Such microneedles may be used for low flow rate, continuous drug delivery, such as the continuous delivery of insulin to a diabetic patient. The needles would be integrated into a short term drug delivery device capable of delivering therapeutics intradermally for about 24 hours. In addition, microneedles can be used for sample collection for biological analysis, delivery of cell or cellular extract based vaccines, and sample handling providing interconnection between the microscopic and macroscopic world.The strength of microneedles was examined analytically, experimentally and by finite element analysis. Metal coatings provide significant increases in the achievable bending moments before failure in the needles. For example, a 10 μ m platinum coating increased the median bending moment of a 160 μ m wide, 110 μ m high microneedle with a 20 μ m wall from 0.25 to 0.43 mNm. In addition, fluid flow in microneedles was studied experimentally. Microneedles 192 μ m wide, 110 μ m high and 7 mm long have flow rates of 0.7 ml/sec under a 138 kPa inlet pressure. This flow capacity exceeds previous microneedle capacities by an order of magnitude.

Cell-Shape Regulation of Smooth Muscle Cell Proliferation

Vascular smooth muscle cells (SMCs) play an important role in vascular remodeling. Heterogeneity and phenotypic changes in SMCs are usually accompanied by a morphological difference, i.e., elongated/spindle-like versus spread-out or epithelioid/rhomboid cell shapes. However, it is not known whether the cell shape directly regulates SMC proliferation, and what the underlying mechanisms are. In this study, microgrooves and micropatterned matrix islands were used to engineer the cell shape and investigate the associated biophysical and biological mechanisms. Compared to spread-out SMCs on nonpatterned surfaces, SMCs on micropatterned surfaces demonstrated elongated morphology, significantly lower cell and nucleus shape indexes, less spreading, a lower proliferation rate, and a similar response (but to a lesser extent) to platelet-derived growth factor, transforming growth factor-beta, and mechanical stretching. DNA microarray profiling revealed a lower expression of neuron-derived orphan receptor-1 (NOR-1) in elongated SMCs. Knocking down NOR-1 suppressed DNA synthesis in SMCs, suggesting that NOR-1 is a mediator of cell elongation effects. Regulation of DNA synthesis in SMCs by the cell shape alone and a decrease in DNA synthesis in the case of small cell spreading area were achieved by micropatterning SMCs on matrix islands of different shapes and spreading areas. Changes in the cell shape also affected the nucleus shape, whereas variations in the cell spreading area modulated the nucleus volume, indicating a possible link between nucleus morphology (both shape and volume) and DNA synthesis. The findings of this investigation provide insight into cell shape effects on cell structure and proliferation, and have direct implications for vascular pathophysiology.

Microneedles and transdermal applications

With the limitations of oral drug delivery and the pain and needle phobias associated with traditional injections, drug delivery research has focused on the transdermal delivery route. A formidable barrier to transdermal drug delivery is the stratum corneum, the superficial layer of the skin. In the last 10 years, microneedles were proposed as a mechanical tool to pierce through the stratum corneum, in order to create drug delivery channels without stimulating underlying pain nerves. Since then, the field of microneedles has rapidly evolved to spawn a plethora of potential transdermal applications. In this review, the authors provide an overview of the progress in microneedle research and design, and the advancements that have been made in employing this technology for transdermal applications.

Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

ABSTRACT There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as thep-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at highp-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 andEscherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.

Continuous On-Chip Micropumping for Microneedle Enhanced Drug Delivery

Microneedles are promising microfabricated devices for minimally invasive drug delivery applications. Needles can be integrated into a variety of devices. However, any portable drug delivery device with integrated microneedles will need an equally compact means to deliver therapeutics. This work presents microneedles integrated with an on-chip MEMS positive displacement micropump for continuous drug delivery applications. The generation and collapse of thermally generated bubbles with flow rectified by directional check valves are used to achieve net pumping through the device. Visualization methods have observed net flow rates of water out of a microneedle at approximately 2.0 nl/s with a pressure of 3.9 kPa. In addition, continuous pumping was achieved for more than 6 hours with the heaters actuating for over 18 hours (15,000 cycles) without failing.

In vivo evaluation of a microneedle-based miniature syringe for intradermal drug delivery

A microfabrication process for miniature syringes is described. The MEMS syringes consist of a silicon plate with an array of hollow out-of-plane needles and a flexible poly-dimethylsiloxane (PDMS) reservoir attached to the back of the plate. The PDMS reservoir can be filled with a drug solution or microparticle suspension which is delivered into the skin simply by the pressure of a finger pushing on the miniature syringe. The efficiency of such a syringe for delivering a suspension of microparticles into skin tissue and a radiolabelled protein (albumin) solution into live mice is reported. Such microneedle devices could be used for the intradermal delivery of vaccination agents or for the systemic delivery of highly effective drugs.