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Publication Hydrostatic Pressure Differentially Regulates Outer and Inner Annulus Fibrosus Cell Matrix Production in 3D Scaffolds(2007-11-17) Reza, Anna T; Nicoll, Steven BMechanical stimulation may be used to enhance the development of engineered constructs for the replacement of load bearing tissues, such as the intervertebral disc. This study examined the effects of dynamic hydrostatic pressure (HP) on outer and inner annulus (OA, IA) fibrosus cells seeded on fibrous poly(glycolic acid)-poly(L-lactic acid) scaffolds. Constructs were pressurized (5 MPa, 0.5 Hz) for four hours/day from day 3 to day 14 of culture and analyzed using ELISAs and immunohistochemistry (IHC) to assess extracellular matrix (ECM) production. Both cell types were viable, with OA cells exhibiting more infiltration into the scaffold, which was enhanced by HP. ELISA analyses revealed that HP had no effect on type I collagen production while a significant increase in type II collagen (COL II) was measured in pressurized OA constructs compared to day 14 unloaded controls. Both OA and IA dynamically loaded scaffolds exhibited more uniform COL II elaboration as shown by IHC analyses, which was most pronounced in OA-seeded scaffolds. Overall, HP resulted in enhanced ECM elaboration and organization by OA-seeded constructs, while IA-seeded scaffolds were less responsive. As such, hydrostatic pressurization may be beneficial in annulus fibrosus tissue engineering when applied in concert with an appropriate cell source and scaffold material.Publication Transient Cervical Nerve Root Compression Modulates Pain: Load Thresholds for Allodynia and Sustained Changes in Spinal Neuropeptide Expression(2007-10-30) Hubbard, Raymond D; Chen, Zen; Winkelstein, Beth ANerve root compression produces chronic pain and altered spinal neuropeptide expression. This study utilized controlled transient loading in a rat model of painful cervical nerve root compression to investigate the dependence of mechanical allodynia on load magnitude. Injury loads (0–110 mN) were applied quasistatically using a customized loading device, and load thresholds to produce maintained mechanical allodynia were defined. Bilateral spinal expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was assessed 7 days following compression using immunohistochemistry to determine relationships between these neuropeptides and compression load. A three-segment change point model was implemented to model allodynia responses and their relationship to load. Load thresholds were defined at which ipsilateral and contralateral allodynia were produced and sustained. The threshold for increased allodynia was lowest for acute (day 1) ipsilateral responses (26.29 mN), while thresholds for allodynia on day 7 were similar for the ipsilateral (38.16 mN) and contralateral forepaw (38.26 mN). CGRP, but not SP, significantly decreased with load; the thresholds for ipsilateral and contralateral CGRP decreases corresponded to 19.52 and 24.03 mN, respectively. These thresholds suggest bilateral allodynia may be mediated by spinal mechanisms, and that these mechanisms depend on the magnitude of load.Publication Photoacoustic effect for multiply scattered light(2007-09-25) Fisher, Andrew R; Schissler, Andrew J; Schotland, John CWe consider the photoacoustic effect for multiply scattered light in a random medium. Within the accuracy of the diffusion approximation to the radiative transport equation, we present a general analysis of the sensitivity of a photoacoustic wave to the presence of one or more small absorbing objects. Applications to tumor detection by photoacoustic imaging are suggested.Publication Chemical and mechanical nerve root insults induce differential behavioral sensitivity and glial activation that are enhanced in combination(2007-08-28) Rothman, Sarah M; Winkelstein, Beth ABoth chemical irritation and mechanical compression affect radicular pain from disc herniation. However, relative effects of these insults on pain symptoms are unclear. This study investigated chemical and mechanical contributions for painful cervical nerve root injury. Accordingly, the C7 nerve root separately underwent chromic gut exposure, 10gf compression, or their combination. Mechanical allodynia was assessed, and glial reactivity in the C7 spinal cord tissue was assayed at days 1 and 7 by immunohistochemistry using GFAP and OX-42 as markers of astrocytes and microglia, respectively. Both chromic gut irritation and 10gf compression produced ipsilateral increases in allodynia over sham (p<0.048); combining the two insults significantly (p<0.027) increased ipsilateral allodynia compared to either insult alone. Behavioral hypersensitivity was also produced in the contralateral forepaw for all injuries, but only the combined insult was significantly increased over sham (p<0.031). Astrocytic activation was significantly increased over normal (p<0.001) in the ipsilateral dorsal horn at 1 day after either compression or the combined injury. By day 7, GFAP-reactivity was further increased for the combined injury compared to day 1 (p<0.001). In contrast, spinal OX-42 staining was generally variable, with only mild activation at day 1. By day 7 after the combined injury, there were significant (p<0.003) bilateral increases in OX-42 staining over normal. Spinal astrocytic and microglial reactivity follow different patterns after chemical root irritation, compression, and a combined insult. The combination of transient compression and chemical irritation produces sustained bilateral hypersensitivity, sustained ipsilateral spinal astrocytic activation and late onset bilateral spinal microglial activation.Publication Principal Component Analysis of Temporal and Spatial Information for Human Gait Recognition(2005-09-01) Das, Sandhitsu; Lazarewicz, Maciej; Finkel, Leif HPrincipal component analysis was applied to human gait patterns to investigate the role and relative importance of temporal versus spatial features. Datasets consisted of various limb and body angles sampled over increasingly long time intervals. We find that spatial and temporal cues may be useful for different aspects of recognition. Temporal cues contain information that can distinguish the phase of the gait cycle; spatial cues are useful for distinguishing running from walking. PCA and related techniques may be useful for identifying features used by the visual system for recognizing biological motion.Publication Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells(2007-06-20) Gerecht, Sharon; Burdick, Jason A.; Ferreira, Lino S; Townsend, Seth A; Langer, Robert; Vunjak-Novakovic, GordanaControl of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the selfrenewal and differentiation of hESCs.Publication Neuronal Ion-Channel Dynamics in Silicon(2006-05-01) Hynna, Kai M; Boahen, KwabenaWe present a simple silicon circuit for modeling voltage-dependent ion channels found within neural cells, capturing both the gating particle's sigmoidal activation (or inactivation) and the bell-shaped time constant. In its simplest form, our ion-channel analog consists of two MOS transistors and a unity-gain inverter. We present equations describing its nonlinear dynamics and measurements from a chip fabricated in a 0.25 /spl µ/m CMOS process. The channel analog's simplicity allows tens of thousands to be built on a single chip, facilitating the implementation of biologically realistic models of neural computation.Publication Magnetic microposts as an approach to apply forces to living cells(2007-07-14) Sniadecki, Nathan J; Anguelouch, Alexandre; Yang, Michael T; Lamb, Corinne M; Liu, Zhijun; Kirschner, Stuart B; Liu, Yaohua; Reich, Daniel H; Chen, Christopher S.Cells respond to mechanical forces whether applied externally or generated internally via the cytoskeleton. To study the cellular response to forces separately, we applied external forces to cells via microfabricated magnetic posts containing cobalt nanowires interspersed among an array of elastomeric posts, which acted as independent sensors to cellular traction forces. A magnetic field induced torque in the nanowires, which deflected the magnetic posts and imparted force to individual adhesions of cells attached to the array. Using this system, we examined the cellular reaction to applied forces and found that applying a step force led to an increase in local focal adhesion size at the site of application but not at nearby nonmagnetic posts. Focal adhesion recruitment was enhanced further when cells were subjected to multiple force actuations within the same time interval. Recording the traction forces in response to such force stimulation revealed two responses: a sudden loss in contractility that occurred within the first minute of stimulation or a gradual decay in contractility over several minutes. For both types of responses, the subcellular distribution of loss in traction forces was not confined to locations near the actuated micropost, nor uniformly across the whole cell, but instead occurred at discrete locations along the cell periphery. Together, these data reveal an important dynamic biological relationship between external and internal forces and demonstrate the utility of this microfabricated system to explore this interaction. Supporting materials: http://www.pnas.org/cgi/content/full/0611613104/DC1Publication Dynamic Computation in a Recurrent Network of Heterogeneous Silicon Neurons(2006-05-01) Merolla, Paul; Boahen, KwabenaWe describe a neuromorphic chip with a two-layer excitatory-inhibitory recurrent network of that exhibits localized clusters of neural activity. Unlike other recurrent networks, the clusters in our network are pinned to certain locations due to transistor mismatch introduced in fabrication. As described in previous work, our pinned clusters respond selectively to oriented stimuli and the neurons' preferred orientations are distributed similar to the visual cortex. Here we show that orientation computation is rapid when activity alternates between layers (staccato-like), dislodging pinned clusters, which promotes fast cluster diffusion.Publication Silicon Neurons That Phase-Lock(2006-05-01) Wittig, John H; Boahen, KwabenaWe present a silicon neuron with a dynamic, active leak that enables precise spike-timing with respect to a time-varying input signal. Our neuron models the mammalian bushy cell, which enhances the phase-locking of its acoustically driven inputs. Our model enhances phase-locking by up to 38% (quantified by vector strength) across a 60 dB range of acoustic intensities, and up to 22% over a passive leak. Its conductance-based log-domain design yields a compact and efficient circuit, fabricated in 0.25 /spl mu/m CMOS, that is an ideal timing-enhancing component for neuromorphic speech recognition systems.