Adaptation of swallowing hyo-laryngeal kinematics is distinct in oral vs. pharyngeal sensory processing.

Before a bolus is pushed into the pharynx, oral sensory processing is critical for planning movements of the subsequent pharyngeal swallow, including hyoid bone and laryngeal (hyo-laryngeal) kinematics. However, oral and pharyngeal sensory processing for hyo-laryngeal kinematics is not fully understood. In 11 healthy adults, we examined changes in kinematics with sensory adaptation, sensitivity shifting, with oropharyngeal swallows vs. pharyngeal swallows (no oral processing), and with various bolus volumes and tastes. Only pharyngeal swallows showed sensory adaptation (gradual changes in kinematics with repeated exposure to the same bolus). Conversely, only oropharyngeal swallows distinguished volume differences, whereas pharyngeal swallows did not. No taste effects were observed for either swallow type. The hyo-laryngeal kinematics were very similar between oropharyngeal swallows and pharyngeal swallows with a comparable bolus. Sensitivity shifting (changing sensory threshold for a small bolus when it immediately follows several very large boluses) was not observed in pharyngeal or oropharyngeal swallowing. These findings indicate that once oral sensory processing has set a motor program for a specific kind of bolus (i.e., 5 ml water), hyo-laryngeal movements are already highly standardized and optimized, showing no shifting or adaptation regardless of repeated exposure (sensory adaptation) or previous sensory experiences (sensitivity shifting). Also, the oral cavity is highly specialized for differentiating certain properties of a bolus (volume) that might require a specific motor plan to ensure swallowing safety, whereas the pharyngeal cavity does not make the same distinctions. Pharyngeal sensory processing might not be able to adjust motor plans created by the oral cavity once the swallow has already been triggered.

Insect-machine interface: a carbon nanotube-enhanced flexible neural probe.

We developed microfabricated flexible neural probes (FNPs) to provide a bi-directional electrical link to the moth Manduca sexta. These FNPs can deliver electrical stimuli to, and capture neural activity from, the insect's central nervous system. They are comprised of two layers of polyimide with gold sandwiched in between in a split-ring geometry that incorporates the bi-cylindrical anatomical structure of the insect's ventral nerve cord. The FNPs provide consistent left and right abdominal stimulation both across animals and within an individual animal. The features of the stimulation (direction, threshold charge) are aligned with anatomical features of the moth. We also have used these FNPs to record neuronal activity in the ventral nerve cord of the moth. Finally, by integrating carbon nanotube (CNT)-Au nanocomposites into the FNPs we have reduced the interfacial impedance between the probe and the neural tissue, thus reducing the magnitude of stimulation voltage. This in turn allows use of the FNPs with a wireless stimulator, enabling stimulation and flight biasing of freely flying moths. Together, these FNPs present a potent new platform for manipulating and measuring the neural circuitry of insects, and for other nerves in humans and other animals with similar dimensions as the ventral nerve cord of the moth.

Encapsulation of ePTFE in prevascularized collagen leads to peri-implant vascularization with reduced inflammation.

During the typical healing response to an implanted biomaterial, vascular-rich granulation tissue forms around the implant and later resolves into a relatively avascular, fibrous capsule. We have previously shown that a microvascular construct (MVC) consisting of isolated microvessel fragments suspended in a collagen I gel forms a persistent microcirculation in lieu of avascular scar when implanted. The current study evaluated the potential for microvascular constructs to maintain a vascularized tissue environment around an implanted biomaterial. An analysis of the peri-implant tissue around bare expanded polytetrafluoroethylene (ePTFE), ePTFE embedded within a microvascular construct, or ePTFE embedded within collagen alone revealed that the presence of the MVC, but not collagen alone, promoted vascular densities comparable to that of the granulation tissue formed around bare ePTFE. The vessels within the microvascular construct surrounding the ePTFE were perfusion competent, as determined by India ink perfusion casting, and extended into the interstices of the polymer. In contrast to bare ePTFE, the presence of the MVC or collagen alone significantly reduced the number of activated macrophages in association with ePTFE. Similar results were observed for ePTFE modified to increase cellularity and prevent the formation of an avascular scar. The microvascular construct may prove effective in forming vascularized tissue environments and limiting the number of activated macrophages around implanted polymers thereby leading to effective implant incorporation.

Flexible split-ring electrode for insect flight biasing using multisite neural stimulation.

We describe a flexible multisite microelectrode for insect flight biasing using neural stimulation. The electrode is made of two layers of polyimide (PI) with gold sandwiched in between in a split-ring geometry. The split-ring design in conjunction with the flexibility of the PI allows for a simple insertion process and provides good attachment between the electrode and ventral nerve cord of the insect. Stimulation sites are located at the ends of protruding tips that are circularly distributed inside the split-ring structure. These protruding tips penetrate into the connective tissue surrounding the nerve cord. We have been able to insert the electrode into pupae of the giant sphinx moth Manduca sexta as early as seven days before the adult moth emerges, and we are able to use the multisite electrode to deliver electrical stimuli that evoke multidirectional, graded abdominal motions in both pupae and adult moths. Finally, in loosely tethered flight, we have used stimulation through the flexible microelectrodes to alter the abdominal angle, thus causing the flying moth to deviate to the left or right of its intended path.

An islet-stabilizing implant constructed using a preformed vasculature.

Islet transplantation for the purpose of treating insulin-sensitive diabetes is currently limited by several factors, including islet survival posttransplantation. In the current study, a tissue-engineered prevascularized pancreatic encapsulating device (PPED) was developed. Isolated islets were placed in collagen gels, and they exhibited fourfold more insulin release than islets not in collagen. The insulin released by beta-cells in islets encapsulated in collagen exhibited unobstructed diffusion within the collagen gels. Subsequent studies evaluated the ability to create a sandwich comprised of two layers of prevascularized collagen gels around a central collagen gel containing islets. In vitro characterization of the islets showed that islets are functional and responded to glucose stimulation. The PPEDs were implanted subcutaneously into severe combined immunodeficient mice. Islet survival was assessed after 7, 14, and 28 days. Immunohistochemical analysis was performed on the implants to detect insulin and the presence of intraislet endothelial cells. At all time points, insulin was localized in association with intact and partially dissociated islets. Moreover, cells that exhibited insulin staining were colocalized with intraislet endothelial cells. These data indicate that the PPED enhances islet survival by supporting islet viability and maintaining intraislet endothelial cell structures.

Texture analysis of speckle in optical coherence tomography images of tissue phantoms.

Optical coherence tomography (OCT) is an imaging modality capable of acquiring cross-sectional images of tissue using back-reflected light. Conventional OCT images have a resolution of 10-15 microm, and are thus best suited for visualizing tissue layers and structures. OCT images of collagen (with and without endothelial cells) have no resolvable features and may appear to simply show an exponential decrease in intensity with depth. However, examination of these images reveals that they display a characteristic repetitive structure due to speckle. The purpose of this study is to evaluate the application of statistical and spectral texture analysis techniques for differentiating living and non-living tissue phantoms containing various sizes and distributions of scatterers based on speckle content in OCT images. Statistically significant differences between texture parameters and excellent classification rates were obtained when comparing various endothelial cell concentrations ranging from 0 cells/ml to 25 million cells/ml. Statistically significant results and excellent classification rates were also obtained using various sizes of microspheres with concentrations ranging from 0 microspheres/ml to 500 million microspheres/ml. This study has shown that texture analysis of OCT images may be capable of differentiating tissue phantoms containing various sizes and distributions of scatterers.

Stimulated endothelial cell adhesion and angiogenesis with laminin-5 modification of expanded polytetrafluoroethylene.

Biomedical implants often exhibit poor clinical performance due to the formation of a periimplant avascular fibrous capsule. Surface modification of synthetic materials has been evaluated to accelerate the formation of functional microcirculation in association with implants. The current study used a flow-mediated protein deposition system to modify expanded polytetrafluoroethylene (ePTFE) with a laminin-5-rich conditioned growth medium and with medium from which laminin-5 had been selectively removed. An in vitro model of endothelial cell adherence determined that laminin-5 modification resulted in significantly increased adhesion of human microvessel endothelial cells to ePTFE. In vivo studies evaluating the periimplant vascular response to laminin-5-treated samples indicated that absorption of laminin-5-rich conditioned medium supported accelerated neovascularization of ePTFE implants. A flow system designed to treat porous implant materials facilitates laminin-5 modification of commercially available ePTFE, resulting in increased endothelial cell adhesion in vitro and increased vascularization in vivo.

Gene expression in tissue associated with extracellular matrix modified ePTFE.

Previous studies have established that surface modification of ePTFE with extracellular matrix molecules promotes vascularization within and around the implanted material. To understand the molecular basis of this tissue response to modified ePTFE, we analyzed large-scale gene expression in nonmodified and extracellular matrix-modified ePTFE-associated healing. Using a microarray containing 15,000 unique mouse cDNAs and an ANOVA-based analysis, we identified 789 genes related to cell signaling, inflammation, matrix remodeling, and proliferation that were differentially expressed across time, between modifications, or both. Genes were clustered based upon similarity in gene expression, producing 7 unique temporal super-patterns of expression. The clustered data revealed 3 general expression patterns unique to tissue surrounding the nonmodified ePTFE, while 6 unique expression patterns were associated with extracellular matrix-modified ePTFE. The more diverse expression patterns associated with extracellular matrix-modified ePTFE suggests that the tissue surrounding the extracellular matrix-modified ePTFE is more dynamic in terms of transcriptional activity. Taken together, these clusters serve as a "genetic fingerprint" for tissue healing in response to a specific material or material modification. Use of these genetic profiles will aid in the pursuit of improved device biocompatibility and enhanced material function.

Three-dimensional bioassembly tool for generating viable tissue-engineered constructs.

The primary emphasis of tissue engineering is the design and fabrication of constructs for the replacement of nonfunctional tissue. Because tissue represents a highly organized interplay of cells and extracellular matrix, the fabrication of replacement tissue should mimic this spatial organization. This report details studies evaluating the use of a three-dimensional, direct-write cell deposition system to construct spatially organized viable structures. A direct-write bioassembly system was designed and fabricated to permit layer-by-layer placement of cells and extracellular matrix on a variety of material substrates. Human fibroblasts suspended in polyoxyethylene/polyoxypropylene were coextruded through a positive displacement pen delivery onto a polystyrene slide. After deposition, approximately 60% of the fibroblasts remained viable. Bovine aortic endothelial cells (BAECs) suspended in soluble collagen type I were coextruded via microdispense pen delivery onto the hydrophilic side of flat sheets of polyethylene terephthalate. After deposition with a 25-gauge tip, approximately 86% of the BAECs were viable. When maintained in culture for up to 35 days, the constructs remained viable and maintained their original spatial organization. These results indicate the potential for utilizing a direct-write, three-dimensional bioassembly tool to create viable, patterned tissue-engineered constructs.