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Stereolithography (SLA) and other photopolymerization-based additive manufacturing approaches are becoming popular for the fabrication of microfluidic devices and cell-infused platforms, but many of the resins employed in these techniques are cytotoxic to cells or do not have the appropriate mechanical properties for microfluidic components. Here, using a commercially available resin, we demonstrate that biocompatibility and a range of mechanical properties can be achieved through post-print optimization involving baking, soaking, network swelling, and UV exposure. We show that UV-vis spectrophotometry can be used to detect methacrylate monomer/oligomer, and utilizing this method, we found that baking at 120 °C for 24 hours was the optimal method for removing cytotoxic chemical species and creating nontoxic cell culture platforms, though UV exposure and soaking in 100% ethanol also can substantially reduce cytotoxicity. Furthermore, we show that the mechanical properties can be modified, including up to 50% for the Young's modulus and an order of magnitude for the flexural modulus, through the post-processing approach employed. Based on the study results, users can choose post-processing approaches to achieve needed cytotoxicity and mechanical profiles, simultaneously.

Introduction: Available evidence suggests that as we age, our brain and immune system undergo changes that increase our susceptibility to injury, inflammation, and neurodegeneration. Since a significant portion of the potential patients treated with a microelectrode-based implant may be older, it is important to understand the recording performance of such devices in an aged population., Methods: We studied the chronic recording performance and the foreign body response (FBR) to a clinically used microelectrode array implanted in the cortex of 18-month-old Sprague Dawley rats., Results and Discussion: To the best of our knowledge, this is the first preclinical study of its type in the older mammalian brain. Here, we show that single-unit recording performance was initially robust then gradually declined over a 12-week period, similar to what has been previously reported using younger adult rats and in clinical trials. In addition, we show that FBR biomarker distribution was similar to what has been previously described for younger adult rats implanted with multi-shank recording arrays in the motor cortex. Using a quantitative immunohistochemcal approach, we observed that the extent of astrogliosis and tissue loss near the recording zone was inversely related to recording performance. A comparison of recording performance with a younger cohort supports the notion that aging, in and of itself, is not a limiting factor for the clinical use of penetrating microelectrode recording arrays for the treatment of certain CNS disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nolta, Christensen and Tresco.)

Gallium (Ga)-based liquid metal materials have emerged as a promising material platform for soft bioelectronics. Unfortunately, Ga has limited biostability and electrochemical performance under physiological conditions, which can hinder the implementation of its use in bioelectronic devices. Here, an effective conductive polymer deposition strategy on the liquid metal surface to improve the biostability and electrochemical performance of Ga-based liquid metals for use under physiological conditions is demonstrated. The conductive polymer [poly(3,4-ethylene dioxythiophene):tetrafluoroborate]-modified liquid metal surface significantly outperforms the liquid metal.based electrode in mechanical, biological, and electrochemical studies. In vivo action potential recordings in behaving nonhuman primate and invertebrate models demonstrate the feasibility of using liquid metal electrodes for high-performance neural recording applications. This is the first demonstration of single-unit neural recording using Ga-based liquid metal bioelectronic devices to date. The results determine that the electrochemical deposition of conductive polymer over liquid metal can improve the material properties of liquid metal electrodes for use under physiological conditions and open numerous design opportunities for next-generation liquid metal-based bioelectronics., (© 2022 Wiley-VCH GmbH.)

Nerve cuffs have been used to anchor and protect penetrating electrodes in peripheral nerves and have been used as non-penetrating electrodes for neural recording and nerve stimulation. The material of choice for such applications is silicone, an inert synthetic biomaterial which elicits a minimal chronic foreign body response (FBR). While histological studies of solid silicone cuffs are available, to the best of our knowledge a comparison to other cuff designs is not well documented. Here, we describe the FBR and morphological changes that accompany nerve cuff implantation in the rat sciatic nerve by comparing a metallic mesh with and without a parylene coating to one made of silicone. Two months after implantation, we observed that such implants, irrespective of the cuff type, were associated with a persistent inflammatory response consisting of activated macrophages attached to the implant surfaces, which extended into the endoneurial space of the encapsulated nerve. We also observed foreign body giant cells in the epineurial space that were more prevalent in the mesh cohorts. The mesh cuff groups showed significant changes in several morphometric parameters that were not seen in the silicon group including reductions in nerve fiber packing density and a greater reduction of large diameter fibers. High magnification microscopy also showed greater evidence of foamy macrophages in the endoneurial space of the mesh implanted cohorts. Although the precise mechanisms are unknown, the results showed that mesh style nerve cuffs show a greater inflammatory response and had greater reductions in morphometric changes in the underlying nerve compared to silicone in the absence of a penetrating injury., Statement of Significance: While traditional silicone cuffs have been in use for decades, the inflammatory and morphometric effects of these cuffs on the underlying nerve have not been deeply studied. Further, manipulation of the foreign body response to nerve cuffs by using various materials and/or designs has not been well reported. Therefore, we report the inflammatory response around nerve cuffs of various materials and designs, as well as report morphometric parameters of the underlying nerve. These data provide important information regarding the potential for quantitative morphometric changes associated with the use of nerve cuffs, and, importantly, suggests that these changes are associated with the degree of inflammation associated with the cuff., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Available evidence suggests that the magnitude of the foreign body response (FBR) to implants placed in cortical brain tissue is affected by the extent of vasculature damage following device insertion and the magnitude of the ensuing macrophage response. Since the extracellular matrix (ECM) serves as a natural hemostatic and immunomodulatory agent, we examined the ability of an FDA-approved neurosurgical hemostatic coating and an ECM coating derived from primary rat astrocytes to reduce the FBR surrounding a penetrating microelectrode array chronically implanted in rat cortex. Using quantitative methods, we examined various components of the FBR in vitro and after implantation. In vitro assays showed that both coatings accelerated coagulation in a similar fashion but only the astrocyte-derived material suppressed macrophage activation. In addition, the ECM coating derived from astrocytes, also decreased the astrogliotic response 8 weeks after implantation. Neither coating had a significant influence on the intensity or spatial distribution of FBR biomarkers 1 week after implantation or on degree of macrophage activation or neuronal survival at the later time point. The results show that microelectrode coatings with similar hemostatic properties but different immunomodulatory characteristics differentially affect the FBR to an anchored, single-shank, silicon microelectrode array. The results also support the concept that divergent biological pathways affect the various components of the FBR in the CNS and suggests that decreasing its impact will require a multifaceted approach., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The surface concentration gradient of two extracellular matrix (ECM) macromolecules was developed to study the migratory and morphological responses of astrocytes to molecular cues typically found in the central nervous system injury environment. The gradient, prepared using microcontact printing, was composed of randomly positioned micrometer-sized dots of aggrecan (AGG) printed on a substrate uniformly coated with laminin (LN). AGG dots were printed in an increasing number along the 1000 μm long and 50 μm wide gradient area which had on each end either a full surface coverage of AGG or LN. Each dot gradient was surrounded by a 100 μm-wide uniform field of AGG printed over laminin. Seeded astrocytes were found to predominantly attach to LN regions on the gradient. Cellular extensions of these cells were longer than the similar processes for cells seeded on uniform substrates of AGG or LN serving as controls. Astrocyte extensions were the largest and spanned a distance of 150 μm when the cells were attached to the mixed AGG+LN patches on the gradient. As evidenced by their increased area and perimeter, the cells extended processes in a stellate fashion upon initial attachment and maintained extensions when seeded in AGG+LN regions but not on uniform laminin controls. The cells migrated short distances, ∼20-35 μm, over 24 h and in doing so preferentially shifted from AGG areas to higher LN surface coverage regions. The results indicated that presenting mixed ECM cues caused astrocytes to sample larger areas of the substrate and made the cells to preferentially relocate to a more permissive ECM region.

The sciatic nerve of rats and cats is commonly used in experimental models of peripheral nerve injury and repair, as well as experiments involving peripheral nerve electrode implantation. In such experiments, morphometric parameters from the implanted nerve are commonly evaluated and compared to control values obtained from the contralateral nerves. However, this may not be an appropriate approach as differences may naturally exist in the structure of the two nerves owing to developmental or behavioral asymmetry. Additionally, in the cat, baseline values for standard morphometric parameters are not well established. In this study, we characterized fascicle area, fiber count, fiber density, fiber packing, mean g-ratio, and fiber diameter distributions in the rat and cat, as well as investigated the potential for naturally occurring sided differences in these parameters in both species. We also investigated whether animal age or location along the nerve influenced these parameters. We found that sided or left/right leg differences exist in some parameters in both the rat and the cat, calling into question the validity of using the contralateral nerve as a control. We also found that animal age and location along the nerve can make significant differences in the parameters tested, establishing the importance of using control nerves from age- and behaviorally matched animals whose morphometric parameters are collected and compared from the same location., (© 2015 Wiley Periodicals, Inc.)

Available evidence indicates that pro-inflammatory cytokines produced by immune cells are likely responsible for the negative sequela associated with the foreign body response (FBR) to chronic indwelling implants in brain tissue. In this study a computational modeling approach was used to design a diffusion sink placed at the device surface that would retain pro-inflammatory cytokines for sufficient time to passively antagonize their impact on the FBR. Using quantitative immunohistochemistry, we examined the FBR to such engineered devices after a 16-week implantation period in the cortex of adult male Sprague-Dawley rats. Our results indicate that thick permeable surface coatings, which served as diffusion sinks, significantly reduced the FBR compared to implants either with no coating or with a thinner coating. The results suggest that increasing surface permeability of solid implanted devices to create a diffusion sink can be used to reduce the FBR and improve biocompatibility of chronic indwelling devices in brain tissue., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The clinical usefulness of brain machine interfaces that employ penetrating silicon microelectrode arrays is limited by inconsistent performance at chronic time points. While it is widely believed that elements of the foreign body response (FBR) contribute to inconsistent single unit recording performance, the relationships between the FBR and recording performance have not been well established. To address this shortfall, we implanted 4X4 Utah Electrode Arrays into the cortex of 28 young adult rats, acquired electrophysiological recordings weekly for up to 12 weeks, used quantitative immunohistochemical methods to examine the intensity and spatial distribution of neural and FBR biomarkers, and examined whether relationships existed between biomarker distribution and recording performance. We observed that the FBR was characterized by persistent inflammation and consisted of typical biomarkers, including presumptive activated macrophages and activated microglia, astrogliosis, and plasma proteins indicative of blood-brain-barrier disruption, as well as general decreases in neuronal process distribution. However, unlike what has been described for recording electrodes that create only a single penetrating injury, substantial brain tissue loss generally in the shape of a pyramidal lesion cavity was observed at the implantation site. Such lesions were also observed in stab wounded animals indicating that the damage was caused by vascular disruption at the time of implantation. Using statistical approaches, we found that blood-brain barrier leakiness and astrogliosis were both associated with reduced recording performance, and that tissue loss was negatively correlated with recording performance. Taken together, our data suggest that a reduction of vascular damage at the time of implantation either by design changes or use of hemostatic coatings coupled to a reduction of chronic inflammatory sequela will likely improve the recording performance of high density intracortical silicon microelectrode arrays over long indwelling periods and lead to enhanced clinical use of this promising technology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As the field of neuroprosthetic research continues to grow, studies describing the foreign body reaction surrounding chronic indwelling electrodes or microelectrode arrays will be critical for assessing biocompatibility. Of particular importance is the reaction surrounding penetrating microelectrodes that are used to stimulate and record from peripheral nerves used for prosthetic control, where such studies on axially penetrating electrodes are limited. Using the Utah Slant Electrode Array and a variety of histological methods, we investigated the foreign body response to the implanted array and its surrounding silicone cuff over long indwelling periods in the cat sciatic nerve. We observed that implanted nerves were associated with increased numbers of activated macrophages at the implant site, as well as distal to the implant, at all time points examined, with the longest observation being 350 days after implantation. We found that implanted cat sciatic nerves undergo a compensatory regenerative response after the initial injury that is accompanied by shifts in nerve fiber composition toward nerve fibers of smaller diameter and evidence of axons growing around microelectrode shafts. Nerve fibers located in fascicles that were not penetrated by the array or were located more than a few hundred microns from the implant appeared normal when examined over the course of a year-long indwelling period., (Copyright © 2014. Published by Elsevier Ltd.)

The collection of cell-derived extracellular matrix (ECM) to form implantable biomaterials has therapeutic potential. However, a significant challenge to the creation of these biomaterials is the ability to produce an adequate quantity of ECM from cells in culture. Mechanical stimulation has long been viewed as a practical means to enhance cellular matrix production. In this study we explored the influence of vocally inspired mechanical stimulation, a unique combination of high frequency vibration and low frequency strain, on the production of ECM. Using a custom fabricated vocal bioreactor, tracheal fibroblast seeded sacrificial foams were treated for 3 weeks using either isolated cyclic strain, combined cyclic strain and vibration (dual mode), or static conditioning. When compared to static controls, ECM production was significantly increased for samples conditioned with either cyclic strain or dual mode stimulation. The quantity of ECM harvested from sacrificial foams increased from 25 ± 1 mg for statically conditioned control foams, to 34 ± 3 and 52 ± 10 mg for cyclic strain and dual mode conditioned samples respectively. Furthermore, mechanical conditioning significantly increased the elastic modulus of ECM biomaterials collected from sacrificial foams. Static control modulus increased from 40 ± 2 to 63 ± 7 kPa and 92 ± 7 kPa following isolated cyclic strain and dual mode conditioning, respectively. These results indicate that cyclic strain conditioning can be used to accelerate the production of ECM by human tracheal cells during growth in culture, and that the addition of high frequency vibration to the conditioning program further enhances ECM production.

Surface-adsorbed fibrinogen (FBG) was recognized by adhering astrocytes, and was removed from the substrates in vitro by a two-phase removal process. The cells removed adsorbed FBG from binary proteins' surface patterns (FBG+laminin, or FBG+albumin) while leaving the other protein behind. Astrocytes preferentially expressed chondroitin sulfate proteoglycan (CSPG) at the loci of fibrinogen stimuli; however, no differences in overall CSPG production as a function of FBG surface coverage were identified. Removal of FBG by astrocytes was also found to be independent of transforming growth factor type β (TGF-β) receptor based signaling as cells maintained CSPG production in the presence of TGF-β receptor kinase inhibitor, SB 431542. The inhibitor decreased CSPG expression, but did not abolish it entirely. Because blood contact and subsequent FBG adsorption are unavoidable in neural implantations, the results indicate that implant-adsorbed FBG may contribute to reactive astrogliosis around the implant as astrocytes specifically recognize adsorbed FBG., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

We have recently reported on a bench-top approach for isolating extracellular matrix (ECM) from pure populations of cells grown in culture using sacrificial, open-celled foams to concentrate and capture the ECM. To increase both the accumulation and the strength of the ECM harvested, cell-seeded polyurethane (PU) foams were cultured in media supplemented with either transforming growth factor β-1 (TGFβ1) or hepatocyte growth factor (HGF). At the end of a 3-week culture period, ECM yield was significantly increased for samples conditioned in supplemented media. Control foams yielded 48 ± 12 mg of material for every gram of PU foam seeded. Yield values increased to 102 ± 21 and 243 ± 25 mg for HGF and TGFβ1-treated samples, respectively. HGF supplementation increased the modulus by 59%, while TGFβ1 treatment increased the elastic modulus by 204%. TGFβ1-stimulated material was organized into a network that was markedly denser than control material, with HGF-stimulated network density intermediate to TGFβ1 and controls. Our study showed that TGFβ1-treated samples were collagen enriched while HGF samples had an increased gylcosaminoglycan concentration. The results demonstrate that growth factor supplementation, particularly with TGFβ1, can significantly alter the biomechanical properties of cell-derived ECM that may be used for therapeutic applications., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteoglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density. DRGs responded by upregulating integrin expression in a proteoglycan dose dependent fashion and exhibited robust outgrowth over all proteoglycan densities at initial time frames. However, after prolonged proteoglycan exposure, neurons exhibited decreasing velocities associated with increasing proteoglycan densities, while neurons growing on low proteoglycan levels exhibited robust outgrowth at all time points. Additionally, DRG outgrowth over proteoglycan density step boundaries, and a brief β1 integrin functional block proved that regeneration was integrin dependent and that DRGs exhibit delayed slowing and loss in persistence after even transient encounters with dense proteoglycan boundaries. These findings demonstrate the complexity of proteoglycan regulation on integrin expression and regenerative pathfinding., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Planar substrates with patterned ligands were used to induce astrocyte alignment whereas substrates with uniform fields of ligand were used to produce random cell orientation. DRG neurons plated on top of oriented astrocyte monolayers exhibited directional outgrowth along aligned astrocytes, demonstrating that purely biological cues provided by the oriented astrocytes were sufficient to provide guidance cues. Antibody blocking studies demonstrated that astrocyte associated FN played a major mechanistic role in directing engineered neurite extension. Our results show that nanometer level surface cues are sufficient to direct nerve outgrowth through an intervening organized astrocyte cell layer. In other studies, we showed that patterned ligands were able to transmit organization cues through multiple cell layers to control the overall alignment of an astrocyte tissue construct, demonstrating how natural scar tissue may develop in situ into potent barriers. In such constructs the spatial organization of astrocyte derived FN maintained its organizational anisotropy throughout the thickness of multilayered astrocyte constructs. These in vitro studies suggest possible roles for such constructs as bridging substrates for neuroregenerative applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Adeno-associated virus (AAV) vector-mediated transgene expression is a promising therapeutic to change the intrinsic state of neurons and promote repair after central nervous system injury. Given that numerous transgenes have been identified as potential candidates, the present study demonstrates how to determine whether their expression by AAV has a direct intrinsic effect on axon regeneration., Methods: Serotype 2 AAV-enhanced green fluorescent protein (EGFP) was stereotaxically injected into the brainstem of adult rats, followed by a complete transection of the thoracic spinal cord and Schwann cell (SC) bridge implantation., Results: The expression of EGFP in brainstem neurons labeled numerous axons in the thoracic spinal cord and that regenerated into the SC bridge. The number of EGFP-labeled axons rostral to the bridge directly correlated with the number of EGFP-labeled axons that regenerated into the bridge. Animals with a greater number of EGFP-labeled axons rostral to the bridge exhibited an increased percentage of those axons found near the distal end of the bridge compared to animals with a lesser number. This suggested that EGFP may accumulate distally in the axon with time, enabling easier visualization. By labeling brainstem axons with EGFP before injury, numerous axon remnants undergoing Wallerian degeneration may be identified distal to the complete transection up to 6 weeks after injury., Conclusions: Serotype 2 AAV-EGFP enabled easy visualization of brainstem axon regeneration. Rigorous models of axonal injury (i.e. complete transection and cell implantation) should be used in combination with AAV-EGFP to directly assess AAV-mediated expression of therapeutic transgenes as intrinsic treatments to improve axonal regeneration., (Copyright © 2012 John Wiley & Sons, Ltd.)

A consistent feature of the foreign body response (FBR), irrespective of the type of implant, is persistent inflammation at the biotic-abiotic interface signaled by biomarkers of macrophage/microglial activation. Since macrophage-secreted factors shape the foreign body reaction, implant designs that reduce macrophage activation should improve biocompatibility and, with regard to recording devices, should improve reliability and longevity. At present, it is unclear whether the goal of seamless integration is possible or whether electrode developers can modulate specific aspects of the FBR by intentionally manipulating the constitutive properties of the implant. To explore this area, we studied the chronic brain FBR to planar solid silicon microelectrode arrays and planar lattice arrays with identical penetrating profiles but with reduced surface area in rats after an 8-week indwelling period. Using quantitative immunohistochemistry, we found that presenting less surface area after equivalent iatrogenic injury is accompanied by significantly less persistent macrophage activation, decreased blood brain barrier leakiness, and reduced neuronal cell loss. Our findings show that it is possible for implant developers to modulate specific aspects of the FBR by intentionally manipulating the constitutive properties of the implant. Our results also support the theory that the FBR to implanted electrode arrays, and likely other implants, can be explained by the presence of macrophages at the biotic-abiotic interface, which act as a sustained delivery source of bioactive agents that diffuse into the adjacent tissue and shape various features of the brain FBR. Further, our findings suggest that one method to improve the recording consistency and lifetime of implanted microelectrode arrays is to design implants that reduce the amount of macrophage activation at the biotic-abiotic interface and/or enhance the clearance or impact of their released factors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Implanted biomedical devices are playing an increasingly important role in the treatment of central nervous system disorders. While devices such as deep brain stimulation electrodes and drug delivery systems have shown clinical success in chronic applications, other devices such as nerve guidance substrates and recording electrodes that operate over a very short length scale have not had the same kind of clinical impact. By reviewing what is currently known about the brain tissue response to implanted electrodes, the authors propose that the foreign-body response, which changes the tissue structure immediately surrounding implanted devices, may be the reason near-function devices are stalled in preclinical development. The article concludes by reviewing recent efforts to reduce the foreign body response, which shows promise to accelerate the clinical development of this new generation of biomedical devices.

Craniofacial reconstruction would benefit from a degradable adhesive capable of holding bone fragments in three-dimensional alignment and gradually being replaced by new bone without loss of alignment or volume changes. Modeled after a natural adhesive secreted by the sandcastle worm, we studied the biocompatibility of adhesive complex coacervates in vitro and in vivo with two different rat calvarial models. We found that the adhesive was non-cytotoxic and supported the attachment, spreading, and migration of a commonly used osteoblastic cell line over the course of several days. In animal studies we found that the adhesive was capable of maintaining three-dimensional bone alignment in freely moving rats over a 12 week indwelling period. Histological evidence indicated that the adhesive was gradually resorbed and replaced by new bone that became lamellar across the defect without loss of alignment, changes in volume, or changes in the adjacent uninjured bone. The presence of inflammatory cells was consistent with what has been reported with other craniofacial fixation methods including metal plates, screws, tacks, calcium phosphate cements and cyanoacrylate adhesives. Collectively, the results suggest that the new bioadhesive formulation is degradable, osteoconductive and appears suitable for use in the reconstruction of craniofacial fractures., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In this study we employed a quantitative immunohistochemical approach to compare the brain tissue response to planar silicon microelectrode arrays that were conformally coated with Parylene-C to uncoated controls at 2, 4, and 12 weeks following implantation into the cortex of adult male Sprague-Dawley rats. We did not find any difference in the relative intensity or the spatial distribution of neuronal or glial markers over the indwelling period, even though Parylene-C-coated substrates supported significantly less cell attachment, indicating that the foreign body response to planar silicon microelectrode arrays has little to do with the composition or decomposition of the silicon electrode. Moreover, our results suggest that changes in microelectrode surface chemistry do not have a strong influence on the cytoarchitectural changes that accompany the brain foreign body response to planar silicon microelectrode arrays. Our quantitative comparison over the indwelling period does not support progressive increases in astrocyte encapsulation and/or progressive neuronal loss in the recording zone as dominant failure mechanisms of the type of chronic recording device. Finally, we found evidence of two potentially new failure mechanisms that were associated with CD68 immunoreactivity including demyelination of adjacent neurons and BBB breakdown surrounding implanted electrodes at long indwelling times., (Copyright © 2010. Published by Elsevier Ltd.)

Extracellular matrix derived from human and animal tissues is being used to repair and reconstruct a variety of tissues clinically. The utility of such constructs is limited by the geometry, composition and constitutive properties of the tissue or organ from which the ECM is harvested. To address this limitation, we have developed an approach to isolate extracellular matrix in bulk from populations of living cells grown in culture on three-dimensional substrates. Human biopsy derived fibroblasts were seeded within open-cell foams and cultured in-vitro for periods up to three weeks, after which the synthetic component was removed by incubation in a water miscible solvent. After several wash steps and lyophilization, a white, lacy, multi-molecular construct was isolated. Tandem mass spectroscopy showed that it contained 22 extracellular matrix constituents, including such proteins and proteoglycans as collagen type I and type III, fibronectin, transforming growth factor beta, decorin and biglycan among others. On average 47 mg of construct was isolated for each gram of synthetic substrate initially seeded with cells. The biomaterial harvested from human tracheal fibroblasts had an elastic modulus (250 kPa) and a composition similar to that of human vocal fold tissue, and supported reseeding with human tracheal derived fibroblasts. An important finding was that the approach was useful in isolating ECM from a variety of cell lineages and developmental stages including skin fibroblasts, brain derived astrocytes and mesenchymal stem cells. The results, together with the archival literature, suggest that the approach can be used to produce a range of cell derived constructs with unique physical and chemical attributes for a variety of research and medical applications., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Several hypotheses have been proposed to explain how the brain tissue reaction to single unit recording electrodes influences biocompatibility including progressive changes in the spatial distribution of reactive astrocytes, and the loss of neurons over the indwelling period. To examine these hypotheses, the spatial distribution of biomarkers associated with the foreign body response to insulated microwires placed in rat cerebral cortex was analyzed 2, 4, and 12 weeks after implantation using quantitative methods. We observed a stereotypical tissue response that was similar in some aspects to that previously reported for penetrating planar silicon microelectrode arrays with some specific differences including an overall lower degree of cortical tissue reactivity. While we found no evidence that reactive gliosis increases over time or that neuronal loss is progressive, we did find evidence of persistent inflammation and enhanced BBB permeability at the electrode brain tissue interface that extended over the 3 month indwelling period and that exhibited more animal to animal variability at 3 months than at 2 and 4 weeks., ((c) 2009 Elsevier Ltd. All rights reserved.)

To study the affect of mechanical stimuli on human laryngeal fibroblasts, we developed bioreactors capable of vibrating cell seeded substrates at frequencies and displacements comparable to measured phonation values in human subjects. In addition, we developed a means of harvesting the secreted matrix as a bulk biomaterial by removing the polymer foam using an organic solvent. Using the system human derived laryngeal fibroblasts were subjected to vibrational stimuli (100 Hz) for 1-21 days. Following mechanical conditioning, extracellular matrix and matrix related gene expression, cytokine production, matrix protein accumulation, and construct material properties were assessed with DNA microarray, enzyme linked immunosorbent, indirect immunofluorescent, and uni-axial tensile assays respectively. The results show that vocal fold-like vibrational stimuli is sufficient to influence the expression of several key matrix and matrix related genes, enhance the secretion of the profibrotic cytokine TGFbeta1, increase the accumulation of the extracellular matrix proteins, fibronectin and collagen type 1, as well as enhance construct stiffness compared to non-stimulated controls. Our results demonstrate that high frequency substrate vibration, like cyclic strain, can accelerate matrix deposition from human derived laryngeal fibroblasts. The study supports the notion that preconditioning regimens using human cells may be useful for producing cell derived biomaterials for therapeutic application.

Proteoglycan expression patterns in the central nervous system guide neuronal pathfinding during development, but also disrupt regeneration after injuries. To deepen our understanding of the molecular level effects of proteoglycan spatial arrangements on neuronal pathfinding, we designed micropatterning stamps for the precise placement of near single molecule chondroitin sulfate proteoglycan (CSPG) clusters into regularly spaced arrays. Actin ultrastructural analysis in dorsal root ganglion neurons grown on laminin-coated substrata patterned with aggrecan cluster arrays revealed filopodial and lamellapodial edge contact avoidance of individual clusters, while growth cone lamellapodia and central domains were able to span multiple clusters over a range of cluster densities. Total internal reflection fluorescence microscopy interrogation of growth cone substratum morphology further revealed persistence of integrin mediated substratum adhesion and local out-of-plane membrane bending over clusters on the height scale of aggrecan glycosaminoglycan side chains. Direct imaging of cell adhesion molecule CD44 expression in growth cones revealed an aggrecan dose dependent upregulation in CD44 molecules. Evidence of CD44 clustering coinciding with underlying aggrecan molecules implies CSPG-CD44 interactions. The results reveal the limited local repulsive effect of CSPGs on neuronal structures and provide evidence that CD44 upregulation in neurons is affected by local CSPG expression.

Damage to axonal tracts of the central nervous system results in costly and permanent disability. The observations of aborted neurite outgrowth and disorganized scarring in injured central nervous system tissue have motivated the hypothesis that engineered bridging devices might facilitate regeneration. It is thought that both the shape and surface chemistry are important design parameters, however, their relative importance is poorly understood. Previously, we utilized smooth cylindrical surfaces to demonstrate that surfaces designed with directionally varying curvature bias in a stereotyped way postnatal dorsal root ganglion axonal regeneration in the direction of minimum curvature independent of surface chemistry. In the present study, we extend this analysis to include adult dorsal root ganglion neurons and cerebellar granule cells, cell types more representative of the challenge faced clinically. We found that axonal outgrowth of both the adult neuron and the central neuron was less sensitive to substrate curvature than the outgrowth of the postnatal neurons. These differences were quantified by constructing distributions describing the probability of outgrowth for a defined range of surface curvatures. Both the adult neuron and the central neuron exhibited a higher probability of extension in high-curvature directions compared to the postnatal neuron implying that surface geometry may not be as potent a cue in directing the regeneration of these neurons. A microtubule-stabilizing agent enhanced the sensitivity to curvature of the adult neuron, partially reversing the increased probability of growing in a high-curvature direction. The results suggest novel methods to enhance directed neuron regeneration using bridging substrates.

Women-controlled methods for prevention of male-to-female sexual transmission of HIV-1 are urgently needed. Providing inhibitory concentrations of HIV-1 reverse transcriptase inhibitors to impede the replication of the virus in the female genital tissue offers a mechanism for prophylaxis of HIV-1. To this end, an intravaginal ring device that can provide long duration delivery of dapivirine, a nonnucleoside reverse transcriptase inhibitor of HIV-1, was developed utilizing a medical-grade polyether urethane. Monolithic intravaginal rings were fabricated and sustained release with cumulative flux linear with time was demonstrated under sink conditions for a period of 30 days. The release rate was directly proportional to the amount of drug loaded. Another release study conducted for a week utilizing liposome dispersions as sink conditions, to mimic the partitioning of dapivirine into vaginal tissue, also demonstrated release rates constant with time. These results qualify polyether urethanes for development of intravaginal rings for sustained delivery of microbicidal agents., ((c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association)

The clinical usefulness of central nervous system recording electrodes is currently limited by inconsistent long-term performance that is believed to be governed by the brain tissue response to the implant. In this study, we observed persistent macrophage biomarker expression at the biotic-abiotic interface surrounding implanted electrodes over a 12-week indwelling period. Using the cell type-specific marker CD11b to examine the cells attached to electrodes retrieved over the indwelling period, we found that most of the cells were activated microglia, the resident macrophage of brain tissue, indicating that the implanted electrodes behave as a persistent inflammatory stimulus. To determine the potential usefulness of different materials as coatings for implanted electrodes, we examined brain-derived microglial cell attachment and cytokine release on a number of medically relevant materials. Our results suggest that activated microglia attach to many of the materials used as external coatings for electrode manufacture, and likely serve as a source of pro-inflammatory and neurotoxic cytokines that may be responsible for reducing the biocompatibility of such implants. Our results also indicate that low protein-binding coatings may be useful in reducing microglial attachment upon implantation in brain tissue and may provide a means of improving electrode biocompatibility.

The influence of tethering silicon microelectrode arrays on the cortical brain tissue reaction was compared with that of untethered implants placed in the same location by identical means using immunoflourescent methods and cell type specific markers over indwelling periods of 1-4 weeks. Compared with untethered, freely floating implants, tethered microelectrodes elicited significantly greater reactivity to antibodies against ED1 and GFAP over time. Regardless of implantation method or indwelling time, retrieved microelectrodes contained a layer of attached macrophages identified by positive immunoreactivity against ED1. In the tethered condition and in cases where the tissue surrounding untethered implants had the highest levels of ED1+ and GFAP+ immunoreactivity, the neuronal markers for neurofilament 160 and NeuN were reduced. Although the precise mechanisms are unclear, the present study indicates that simply tethering silicon microelectrode arrays to the skull increases the cortical brain tissue response in the recording zone immediately surrounding the microelectrode array, which signals the importance of identifying this important variable when evaluating the tissue response of different device designs, and suggests that untethered or wireless devices may elicit less of a foreign body response., (Copyright 2007 Wiley Periodicals, Inc.)

We present an in vitro micropatterning approach in which the density and spatial presentation of two separate protein layers can be independently controlled to form cell stripe assays through (1) the simultaneous application of microcontact printing (microCP) and microfluidic network (microFN) patterning to generate alternating stripes of pure single protein layers or (2) through microCP onto a pre-adsorbed homogeneous protein layer to generate alternating single and dual protein stripes. This approach enabled the creation of choice boundaries in which protein-protein interactions were limited and the effects of spatially segregated or colocalized dual protein signals on model primary neuronal behavior could be readily interrogated and compared on both glass and tissue culture polystyrene substrates. Dorsal root ganglion (DRG) cell body attachment was dictated largely by non-specific cell adhesion interactions and interactions between the guidance molecules laminin and aggrecan were insufficient to explain aggrecan inhibition on neurite outgrowth. The presentation of a specific laminin epitope stabilized by interactions with aggrecan and destabilized by microCP was a strong predictor of neurite promoting activity. These observations provide evidence that aggrecan is intrinsically inhibitory and that laminin-aggrecan interactions do not diminish laminin growth promoting properties.

The transplantation of genetically engineered fibroblasts has been shown to be an effective approach for achieving continuous and site-specific delivery of therapeutic molecules to various regions of the central nervous system. However, to our knowledge no one has asked whether soluble factors released from the transplanted fibroblasts influence the delivery of therapeutic molecules from the engrafted cells. To address this issue, we used a newly developed cell encapsulation device to study the functional consequence of the foreign body response on soluble factor delivery from fibroblasts transplanted into adult brain tissue. We found that transplanted fibroblasts increased the level of inflammation and glial cell encapsulation at the transplantation site, and reduced the diffusion of a 70 kDa dextran probe through the reactive tissue. The response, however, did not prevent the diffusion of the 70 kDa dextran test probe indicating that the approach appears suitable for the delivery of neurotrophins and other therapeutic molecules with a molecular weight less than 70 kDa. The results suggest that less reactive cell types may be better suited for sustained delivery of therapeutic molecules into brain tissue.

Biodegradable permeable poly(DL-lactide-co-glycolide) (PLGA) hollow fiber membranes (HFMs) were fabricated using a wet phase inversion technique. By varying several parameters, such as the spinneret size, solvent and non-solvent pair, polymer concentration, flow rate, precipitation method, drop height, and small molecular pore-forming agents, PLGA HFMs with variable sizes, surface morphologies, porosities, and diffusive permeability were obtained. Under simulated physiological conditions in vitro, PLGA HFMs exhibited a degradation profile to accommodate nervous system regeneration and axonal outgrowth. While accelerated degradation resulted in substantial molecular weight loss starting at 2 weeks and loss of selective permeability at 3 weeks, PLGA HFMs maintained gross structural integrity in the first 4 weeks, followed by sharp weight loss at 6 weeks and complete disappearance at about 8 weeks. When compared to the raw PLGA material in a pellet form, which underwent heterogeneous degradation, the PLGA HFMs exhibited a homogeneous degradation where the surface and bulk degraded at approximately the same rate, and an overall lower degradation rate. Our results indicate that using a wet phase inversion technique, degradable HFMs with variable size, inner and outer surface morphologies, porosity, and permeability with potential applications for nerve tract guidance conduits can be fabricated.

Object: Researchers at The Center for Neural Interfaces at the University of Utah have designed and produced a silicon-based high-density microelectrode array that has been used successfully in mammalian models. The authors investigate the ability to transfer array insertion techniques to humans and examine the acute response of human cortical tissue to array implantation., Methods: Six patients who were scheduled to undergo temporal lobectomy surgery were enrolled in an Institutional Review Board-approved protocol. Before the patients underwent lateral temporal cortical resection, one or two high-density microelectrode arrays were implanted in each individual by using a pneumatic insertion device. Cortical tissue was then excised and preserved in formalin. The specimens were sectioned and stained for histological examination. Pneumatic insertion of a microelectrode array into human cortex in the operating room was feasible. There were no clinical complications associated with implantation and no evidence of significant insertion-related hemorrhage. Tissue responses ranged from mild cortical deformity to small focal hemorrhages several millimeters below the electrode tines. Based on initial results, the insertion device was modified. A footplate that mechanically isolates a small area of cortex and a calibrated micromanipulator were added to improve the reproducibility of insertion., Conclusions: A high-density microelectrode array designed to function as a direct cortical interface device can be implanted into human cortical tissue without acute clinical complications. Further modifications to the insertion device and array design are ongoing and future work will assess the functional significance of the tissue reactions observed.

At present there is no clinically effective treatment for injuries or pathological processes that disrupt the continuity of axons in the mature central nervous system. However, a number of studies suggest that a tremendous potential exists for developing biomaterial based therapies. In particular, biomaterials in the form of bridging substrates have been shown to support at least some level of axonal regeneration across the lesion site, but display a limited capacity for directing axons toward their targets. To improve the directionality and outgrowth rate of the axonal regeneration process, filaments and tubes appear promising, but the technology is far from optimized. As a step toward optimization, the influence of filament diameter and various extracellular matrix coatings on nerve regeneration was evaluated in this article using a dorsal root ganglion (DRG) explant model. An increasing pattern of alignment and outgrowth of neurites in the direction parallel the long axis of the packed filament bundles with decreasing filament diameters ranging from supracellular and beyond (500 to 100 mum), cellular (30 mum), down to subcellular size (5 mum) was observed. Such effects became most prominent on filament bundles with individual filament diameters in the range of cellular size and below (5 and 30 mum) where highly directional and robust neuronal outgrowth was achieved. In addition, laminin-coated filaments that approached the size of spinal axons support significantly longer regenerative outgrowth than similarly treated filaments of larger diameter, and exceed outgrowth distance on similarly sized filaments treated with fibronectin. These data suggested the feasibility of using a multifilament entubulation bridging device for supporting directional axonal regeneration., ((c) 2005 Wiley Periodicals, Inc.)

Rapid induction of matrix production and mechanical strengthening is essential to the development of bio-artificial constructs for repair and replacement of load-bearing connective tissues. Toward this end, we describe the development of a mechanical bioreactor and its application to investigate the influence of cyclic strain on fibroblast proliferation, matrix accumulation, and the mechanical properties of fibroblast-seeded polyurethane constructs (FSPC). Human fibroblasts were cultured in 10% serum-containing conditions within three-dimensional, porous elastomeric substrates under static conditions and a model regime of cyclic strain (10% strain, 0.25 Hz, 8 h/day), with and without ascorbic acid supplementation. After one week, the combination of cyclic strain and ascorbic acid resulted in significantly increased construct elastic modulus (>110%) relative to either condition alone. In contrast, cyclic strain alone was sufficient to stimulate significant increases in fibroblast proliferation. Mechanical strengthening of FSPCs was accompanied by increased type I collagen and fibronectin matrix accumulation and distribution, and significantly increased gene expression for type I collagen, TGFbeta-1, and CTGF. These results suggest that strain-induced conditioning in vitro leads to mechanical strengthening of fibroblast/material constructs, most likely resulting from increased collagen matrix deposition, secondary to strain-induced increases in cytokine production.

The mechanisms underlying performance degradation of chronically implanted silicon microelectrode arrays in the central nervous system (CNS) remain unclear. Humoral and cellular components of the brain foreign body response were evaluated to determine whether their presence on the electrode surface results in increased electrical impedance. Iridium oxide microelectrode recording arrays were electrically characterized in saline, culture media with 10% fetal bovine serum, and coated with various CNS cell types isolated from rat brain. Electrochemical impedance spectroscopy and cyclic voltammetry were performed using a three-electrode system. Potential cycling caused an immediate decrease in electrical impedance, which increased with time toward precycling values, with the effect of cycling remaining significant for several days. The addition of serum caused a significant increase in impedance of up to 28% relative to the saline control. Microelectrodes coated with various cell types known to participate in the foreign body response caused a 20%-80% increase in impedance immediately after contact that remained constant or gradually increased for several weeks. Our findings suggest that the attachment of molecular and cellular species following microelectrode implantation into brain tissue likely contribute to increases in impedance, but do not appear sufficient to hinder recording performance.

Chronically implanted recording electrode arrays linked to prosthetics have the potential to make positive impacts on patients suffering from full or partial paralysis. Such arrays are implanted into the patient's cortical tissue and record extracellular potentials from nearby neurons, allowing the information encoded by the neuronal discharges to control external devices. While such systems perform well during acute recordings, they often fail to function reliably in clinically relevant chronic settings. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants, making the biocompatibility of implanted electrodes a primary concern in device design. This review presents the biological components and time course of the acute and chronic tissue reaction in brain tissue, analyses the brain tissue response of current electrode systems, and comments on the various material science and bioactive strategies undertaken by electrode designers to enhance electrode performance.

Implantable silicon microelectrode array technology is a useful technique for obtaining high-density, high-spatial resolution sampling of neuronal activity within the brain and holds promise for a wide range of neuroprosthetic applications. One of the limitations of the current technology is inconsistent performance in long-term applications. Although the brain tissue response is believed to be a major cause of performance degradation, the precise mechanisms that lead to failure of recordings are unknown. We observed persistent ED1 immunoreactivity around implanted silicon microelectrode arrays implanted in adult rat cortex that was accompanied by a significant reduction in nerve fiber density and nerve cell bodies in the tissue immediately surrounding the implanted silicon microelectrode arrays. Persistent ED1 up-regulation and neuronal loss was not observed in microelectrode stab controls indicating that the phenotype did not result from the initial mechanical trauma of electrode implantation, but was associated with the foreign body response. In addition, we found that explanted electrodes were covered with ED1/MAC-1 immunoreactive cells and that the cells released MCP-1 and TNF-alpha under serum-free conditions in vitro. Our findings suggest a potential new mechanism for chronic recording failure that involves neuronal cell loss, which we speculate is caused by chronic inflammation at the microelectrode brain tissue interface.

After injury to the CNS, the anatomical organization of the tissue is disrupted, posing a barrier to the regeneration of axons. Meningeal cells, a central participant in the CNS tissue response to injury, migrate into the core of the wound site in an unorganized fashion and deposit a disorganized extracellular matrix (ECM) that produces a nonpermissive environment. Previous work in our laboratory has shown that the presentation of nanometer-scale topographic cues to these cells influences their morphological, cytoskeletal, and secreted ECM alignment. In the present study, we provided similar environmental cues to meningeal cells and examined the ability of the composite construct to influence dorsal root ganglion regeneration in vitro. When grown on control surfaces of meningeal cells lacking underlying topographic cues, there was no bias in neurite outgrowth. In contrast, when grown on monolayers of meningeal cells with underlying nanometer-scale topography, neurite outgrowth length was greater and was directed parallel to the underlying surface topography even though there exists an intervening meningeal cell layer. The observed outgrowth was significantly longer than on laminin-coated surfaces, which are considered to be the optimal substrata for promoting outgrowth of dorsal root ganglion neurons in culture. These results suggest that the nanometer-level surface finish of an implanted biomaterial may be used to organize the encapsulation tissue that accompanies the implantation of materials into the CNS. It furthermore suggests a simple approach for improving bridging materials for repair of nerve tracts or for affecting cellular organization at a device-tissue interface.

Nerve outgrowth in the developing nervous system utilizes a variety of attractive and repulsive molecules found in the extracellular environment. In addition, physical cues may play an important regulatory role in determining directional outgrowth of nervous tissue. Here, by culturing nerve cells on filamentous surfaces and measuring directional growth, we tested the hypothesis that substrate curvature is sufficient to influence the directional outgrowth of nerve cells. We found that the mean direction of neurite outgrowth aligned with the direction of minimum principle curvature, and the spatial variance in outgrowth direction was directly related to the maximum principle curvature. As substrate size approached the size of an axon, adherent neurons extended processes that followed the direction of the long axis of the substrate similar to what occurs during development along pioneering axons and radial glial fibers. A simple Boltzmann model describing the interplay between adhesion and bending stiffness of the nerve process was found to be in close agreement with the data suggesting that cell stiffness and substrate curvature can act together in a manner that is sufficient to direct nerve outgrowth in the absence of contrasting molecular cues. The study highlights the potential importance of cellular level geometry as a fidelity-enhancing cue in the developing and regenerating nervous system.

The transplantation of a variety of naturally occurring and genetically modified cell types has been shown to be an effective experimental method to achieve sustained delivery of therapeutic molecules to specific target areas in the brain. To acquire a better understanding of dosing, implant mechanism of action, and how certain cell types affect remodeling of central nervous system (CNS) tissue, a refillable cell encapsulation device was developed for introducing cells into the brain while keeping them physically isolated from contact with brain tissue with a semipermeable membrane. The stereotactically placed device consists of a hollow fiber membrane (HFM), a polyurethane grommet with watertight cap that snaps into a precisely drilled hole in the rat skull, and a removable cell-containing insert. The cell-containing insert can be introduced or removed in a time-dependent manner to study the influence of soluble factors released from transplanted cells. The study describes the device design and validates its utility using a well-established cell transplantation model of Parkinson's disease.

Objectives: Determine the resorption rate and biocompatibility characteristics of 2 polyester ventilation tubes, and to determine whether soap and water exposure accelerates polyester tube degradation., Study Design and Setting: 50/50 poly (D, L-lactide-co-glycolide; PLGA-50) and poly (L-lactide; PLA) polymers were placed into the tympanic membranes of Hartley pigmented guinea pigs. Integrity of the tubes was determined by weekly otoscopic examination. Biocompatibility was assessed by comparing auditory brainstem response (ABR) thresholds and by examining tympanic membrane changes following tube resorption. Shah minigrommet ventilation tubes were used as controls. In the second portion of this study, implanted PLGA-50 and PLA tubes were exposed weekly to a mixture of soap and water (1:5) until complete resorption was observed. Biocompatibility was assessed by periodic ABR testing and tympanic membrane examination., Results: The PLA tubes remained in the tympanic membrane for a longer period (63.2 +/- 19.3 days) than the PLGA-50 (18.8 +/- 8.1 days). The tympanic membrane and resorbable tube interface demonstrated equivalent findings for auditory thresholds and tissue histopathology at the implant site compared to nonresorbable controls. The resorption behavior was not altered by exposure to soap and water. Tympanic membranes of all animals following tube degradation and soap water exposure were intact with minimal scarring and no signs of persistent foreign body response. The histological analysis showed that implantation of resorbable tubes was not accompanied by secondary infection with otorrhea through the tube, did not result in a permanent perforation or dislocation of the tube into the middle ear cavity, and was not followed by excess tympanosclerosis or localized or diffuse membrane atrophy., Conclusions and Significance: Resorbable polyester pressure equalization tubes demonstrate predictable resorption behavior and similar biocompatibility characteristics when compared with nonresorbable Shah minigrommet ventilation tubes. Exposure to soap water does not accelerate polyester tube degradation nor change the host tissue response during the indwelling period or after complete resorption. The data suggests that resorbable ventilation tubes are substantially equivalent to other FDA-approved tympanostomy devices with regard to safety and biocompatibility in the guinea pig model examined and may provide improved clinical performance by combining this approach with sustained release technology., Ebm Rating: B-2.

It was investigated whether the addition of basic fibroblast growth factor (FGF-2) enhances the efficacy of a Schwann cell (SC) bridge to repair the transected spinal cord by assessing tissue sparing and neuronal survival near the graft-cord interfaces, axonal regeneration and myelination in the graft, and behavioral recovery up to 12 weeks post-grafting. Experimental animals received a bridge of SCs within fibrin containing 1 microg of FGF-2; control animals received a SC implant without FGF-2. Sparing of tissue in a 2.5-mm-long segment near the graft-cord borders was 69% in the rostral and 52% in the caudal cord at 6 weeks post-grafting, not significantly different from the control group. With FGF-2, survival of NeuN-positive cells was increased in the rostral cord: 24.4%, 20.4%, and 17.2% of the number of positive cells in the uninjured cord compared to 13.5%, 9.1%, and 8.9% in controls at 3, 6, and 12 weeks post-grafting, respectively. Similarly, in the caudal cord, survival of NeuN-positive cells was increased with FGF-2: 19.3%, 16.8%, and 14.5% compared to 10.8%, 5.6%, and 6.1% in controls. The staining intensity of glial fibrillary acidic protein was significantly higher at the interfaces of both cord stumps at 3 weeks with SC/FGF-2 grafts; chondroitin sulfate proteoglycan (CS-56) staining was more intense in the rostral cord but only at 6 weeks. Blood vessels in the FGF-2 grafts were larger and less regular in shape than those in control grafts. Axonal growth into the bridge was not improved by the addition of FGF-2. Retrogradely traced neurons were not found rostral to the implant, indicating that axons had not grown a few mm into the caudal spinal tissue. Recovery of hind limb function was similar in both groups. Despite the neuroprotective effects of FGF-2, improved effects on axonal regeneration and functional recovery were not observed.

Criteria are outlined for the design of a bioreactor that can simulate the vibrational stresses in vocal fold movement during speech. Requirements are 0-1 mm amplitudes in the 20-200 Hz frequency range, a variable on-off stress regime, and maintenance of tissue viability over several days. The bioreactor uses dual drivers, one for low frequency (or static) strains, and another for high-frequencies vibrational strains. Response is linear at the driving end for an input of 0-5 V. The amplitude decreases linearly with frequency at constant input voltage, and the phase changes by nearly 180 degrees over the 20-200 Hz range. Human vocal fold fibroblasts were cultured in a polymer substrate and subjected to static and vibrational forces. The results indicate that vibratory strain alters the expression levels of many extracellular matrix-related genes, as well as the spatial distribution of cells and matrix.

The scarring response following injury to the central nervous system disrupts the anatomical organization of nervous tissue posing a barrier to the regeneration of axons. In the present study, using materials with nanometer level surface features we examined whether matrix organization could be controlled by engineering meningeal cell asymmetry. Following 5 days in culture, the organization of meningeal cells along with their cytoskeletal elements and extracellular matrix proteins was evaluated. Meningeal cell morphology was markedly affected by nanometer level substrate topography. Cell alignment increased with increasing surface roughness. In addition, linear arrays of extracellular matrix were expressed that appeared related to cellular orientation. When cultured on substrates with topographical features of less than 10 nm neither cells nor their extracellular matrix showed organizational asymmetry. However, as oriented surface roughness increased, cellular and matrix asymmetrical organization became more pronounced reaching a threshold at 345 nm. These results suggest that biomaterial surface topography or other methods of altering the orientation of cells may be used to engineer orientation into the secreted extracellular matrix and as such may be a potential strategy for developing organized cell-derived matrix as a bridging material for nerve repair or other regenerative applications.

To circumvent the reconstructive disadvantages inherent in resorbable polyglycolic acid (PGA)/polylactic acid (PLA) used in cartilage engineering, a nonresorbable, and nonreactive polyurethane sponge (Tecoflex sponge, TS) was studied as both a cell delivery device and as an internal support scaffolding. The in vitro viability and proliferation of porcine articular chondrocytes (PACs) in TS, and the in vivo generation of new articular cartilage and long-term resorption, were examined. The initial cell attachment rate was 40%, and cell density increased more than 5-fold after 12 days of culture in vitro. PAC-loaded TS blocks were implanted into nude mice, became opalescent, and resembled native cartilage at weeks 12 and 24 postimplantation. The mass and volume of newly formed cartilage were not significantly different at week 24 from samples harvested at week 6 or week 12. Safranin O-fast green staining revealed that the specimens from cell-loaded TS groups at week 12 and week 24 consisted of mature cartilage. Collagen typing revealed that type II collagen was present in all groups of tissue-engineered cartilage. In conclusion, the implantation of PAC-TS resulted in composite tissue-engineered articular cartilage with TS as an internal support. Long-term observation (24 weeks) of mass and volume showed no evidence of resorption.

Using quantitative immunohistological methods, we examined the brain tissue response to hollow fiber membranes (HFMs) that were either implanted intraparenchymally, as in a cell encapsulation application, or were attached to the skull as in a biosensor application (transcranially). We found that the reaction surrounding transcranially implanted HFMs was significantly greater than that observed with intraparenchymally implanted materials including increases in immunoreactivity against GFAP, vimentin, ED-1 labeled macrophages and microglia, and several extracellular matrix proteins including collagen, fibronectin, and laminin. In general, these markers were elevated along the entire length of transcranially implanted HFMs extending into the adjacent parenchyma up to 0.5 mm from the implant interface. Intraparenchymal implants did not appear to have significant involvement of a fibroblastic component as suggested by a decreased expression of vimentin, fibronectin and collagen-type I at the implant tissue interface. The increase in tissue reactivity observed with transcranially implanted HFMs may be influenced by several mechanisms including chronic contact with the meninges and possibly motion of the device within brain tissue. Broadly speaking, our results suggest that any biomaterial, biosensor or device that is anchored to the skull and in chronic contact with meningeal tissue will have a higher level of tissue reactivity than the same material completely implanted within brain tissue.

The mechanisms underlying performance degradation of electrodes chronically implanted in the central nervous system (CNS) remain unclear. Several components of the normal brain wound healing response were evaluated to determine if their presence correlates with increased electrical impedance that may be a factor in loss of device performance. Microelectrode recording arrays were electrically characterized in vitro in the presence of saline, culture media with 10% fetal bovine serum (FBS), and various CNS cell types isolated from Sprague-Dawley rats and cultured in media with 10% FBS. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed using a three-electrode system. Potential cycling during CV caused an immediate decrease in electrical impedance, which increased with time toward the pre-cycling value, with the effect of cycling remaining significant for several days. The addition of serum caused a significant but not substantial increase in impedance. The inclusion of various cell types known to participate in the brain wound healing response caused a significant increase in impedance immediately after seeding on the order of 50%, and this value increased or remained constant for up to several weeks. It is unclear whether the magnitude of increased impedance is sufficient to account for loss of device performance.