Publications by authors named "Ravi V Bellamkonda"

100 Publications

Enriching neural stem cell and anti-inflammatory glial phenotypes with electrical stimulation after traumatic brain injury in male rats.

J Neurosci Res 2021 Jul 26;99(7):1864-1884. Epub 2021 Mar 26.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, USA.

Traumatic brain injury (TBI) by an external physical impact results in compromised brain function via undesired neuronal death. Following the injury, resident and peripheral immune cells, astrocytes, and neural stem cells (NSCs) cooperatively contribute to the recovery of the neuronal function after TBI. However, excessive pro-inflammatory responses of immune cells, and the disappearance of endogenous NSCs at the injury site during the acute phase of TBI, can exacerbate TBI progression leading to incomplete healing. Therefore, positive outcomes may depend on early interventions to control the injury-associated cellular milieu in the early phase of injury. Here, we explore electrical stimulation (ES) of the injury site in a rodent model (male Sprague-Dawley rats) to investigate its overall effect on the constituent brain cell phenotype and composition during the acute phase of TBI. Our data showed that a brief ES for 1 hr on day 2 of TBI promoted anti-inflammatory phenotypes of microglia as assessed by CD206 expression and increased the population of NSCs and Nestin astrocytes at 7 days post-TBI. Also, ES effectively increased the number of viable neurons when compared to the unstimulated control group. Given the salience of microglia and neural stem cells for healing after TBI, our results strongly support the potential benefit of the therapeutic use of ES during the acute phase of TBI to regulate neuroinflammation and to enhance neuroregeneration.
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http://dx.doi.org/10.1002/jnr.24834DOI Listing
July 2021

Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury.

Sci Adv 2021 Mar 5;7(10). Epub 2021 Mar 5.

Regenerative Bioscience Center, University of Georgia, Athens, GA 30602, USA.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain's poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. However, the efficacy of engineered CS (eCS) matrices in mediating structural and functional recovery chronically after sTBI has not been investigated. We report that neurotrophic factor functionalized acellular eCS matrices implanted into the rat M1 region acutely after sTBI significantly enhanced cellular repair and gross motor function recovery when compared to controls 20 weeks after sTBI. Animals subjected to M2 region injuries followed by eCS matrix implantations demonstrated the significant recovery of "reach-to-grasp" function. This was attributed to enhanced volumetric vascularization, activity-regulated cytoskeleton (Arc) protein expression, and perilesional sensorimotor connectivity. These findings indicate that eCS matrices implanted acutely after sTBI can support complex cellular, vascular, and neuronal circuit repair chronically after sTBI.
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http://dx.doi.org/10.1126/sciadv.abe0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935369PMC
March 2021

Immuno-suppressive hydrogels enhance allogeneic MSC survival after transplantation in the injured brain.

Biomaterials 2021 01 28;266:120419. Epub 2020 Sep 28.

Dept. of Biomedical Engineering, Duke University, Durham, NC, 27708, USA. Electronic address:

Traumatic brain injury (TBI) triggers multiple biochemical and cellular processes that exacerbate brain tissue damage through a secondary injury. Therapies that prevent or limit the evolution of secondary injury could significantly reduce the neurological deficits associated with TBI. Mesenchymal stem cell (MSC) transplantation after TBI can ameliorate neurological deficits by modulating inflammation and enhancing the expression of neurotrophic factors. However, transplanted MSCs can be actively rejected by host immune responses, such as those mediated by cytotoxic CD8 T cells, thereby limiting their therapeutic efficacy. Here, we designed an agarose hydrogel that releases Fas ligand (FasL), a protein that can induce apoptosis of cytotoxic CD8 T cells. We studied the immunosuppressive effect of this hydrogel near the allogeneic MSC transplantation site and its impact on the survival of transplanted MSCs in the injured brain. Agarose-FasL hydrogels locally reduced the host cytotoxic CD8 T cell population and enhanced the survival of allogeneic MSCs transplanted near the injury site. Furthermore, the expression of crucial neurotrophic factors was elevated in the injury penumbra, suggesting an enhanced therapeutic effect of MSCs. These results suggest that the development of immunosuppressive hydrogels for stem cell delivery can enhance the benefits of stem cell therapy for TBI.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120419DOI Listing
January 2021

The impact of modulating the blood-brain barrier on the electrophysiological and histological outcomes of intracortical electrodes.

J Neural Eng 2019 08 2;16(4):046005. Epub 2019 May 2.

School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, GA, United States of America.

Objective: Successful application of chronic intracortical electrodes remains highly variable. The biological mechanisms leading to electrode failure are still being explored. Recent work has shown a correlation between blood-brain barrier (BBB) integrity and long-term recordings. Here we proposed to modulate the BBB healing after intracortical electrode implantation, while evaluating the functional electrophysiology. The CCL2/CCR2 pathway was chosen based on previous work demonstrating the positive histological effects in an intracortical electrode model, as well as in other neurodegenerative models. By disrupting this pathway, recruitment of pro-inflammatory monocytes (a result of a breached BBB) is potentially reduced at the electrode interface.

Approach: Michigan electrodes were implanted for 2 and 12 weeks in rats, and a CCR2 antagonist (RS 102895) was administered daily to the treatment group. Functional electrodes were used for the 12 week cohort, and weekly electrophysiological recordings were taken. At 2 and 12 weeks, histology was analyzed.

Main Results: At 12 weeks, the CCR2-antagonist group had significantly higher signal-to-noise ratios (SNRs) than control. CCR2-antagonism at 2 weeks significantly increased the neural population and decreased BBB breach. At 12 weeks, CCR2-antagonism significantly increased number of neurons and BBB  +  vasculature within 100 µm of the electrode interface.

Significance: This work demonstrates that for intracortical electrodes, disruption of the CCL2/CCR2 pathway improves chronic outcomes in electrophysiology and histology.
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http://dx.doi.org/10.1088/1741-2552/ab1ef9DOI Listing
August 2019

Electrotaxis of Glioblastoma and Medulloblastoma Spheroidal Aggregates.

Sci Rep 2019 03 29;9(1):5309. Epub 2019 Mar 29.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 101 Science Drive, Durham, NC, 27705, USA.

Treatment of neuroepithelial cancers remains a daunting clinical challenge, particularly due to an inability to address rampant invasion deep into eloquent regions of the brain. Given the lack of access, and the dispersed nature of brain tumor cells, we explore the possibility of electric fields inducing directed tumor cell migration. In this study we investigate the properties of populations of brain cancer undergoing electrotaxis, a phenomenon whereby cells are directed to migrate under control of an electrical field. We investigate two cell lines for glioblastoma and medulloblastoma (U87mg & DAOY, respectively), plated as spheroidal aggregates in Matrigel-filled electrotaxis channels, and report opposing electrotactic responses. To further understand electrotactic migration of tumor cells, we performed RNA-sequencing for pathway discovery to identify signaling that is differentially affected by the exposure of direct-current electrical fields. Further, using selective pharmacological inhibition assays, focused on the PI3K/mTOR/AKT signaling axis, we validate whether there is a causal relationship to electrotaxis and these mechanisms of action. We find that U87 mg electrotaxis is abolished under pharmacological inhibition of PI3Kγ, mTOR, AKT and ErbB2 signaling, whereas DAOY cell electrotaxis was not attenuated by these or other pathways evaluated.
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http://dx.doi.org/10.1038/s41598-019-41505-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441013PMC
March 2019

Engineering Controlled Peritumoral Inflammation to Constrain Brain Tumor Growth.

Adv Healthc Mater 2019 02 11;8(4):e1801076. Epub 2018 Dec 11.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 101 Science Drive, Durham, NC, 27705, USA.

Brain tumors remain a great clinical challenge, in part due to their capacity to invade into eloquent, inoperable regions of the brain. In contrast, inflammation in the central nervous system (CNS) due to injuries activates microglia and astrocytes culminating in an astroglial scar that typically "walls-off" the injury site. Here, the hypothesis is tested that targeting peritumoral cells surrounding tumors to activate them via an inflammatory stimulus that recapitulates the sequelae of a traumatic CNS injury, could generate an environment that would wall-off and contain invasive tumors in the brain. Gold nanoparticles coated with inflammatory polypeptides to target stromal cells in close vicinity to glioblastoma (GBM) tumors, in order to activate these cells and stimulate stromal CNS inflammation, are engineered. It is reported that this approach significantly contains tumors in rodent models of GBM relative to control treatments (reduction in tumor volume by over 300% in comparison to controls), by the activation of the innate and adaptive immune response, and by triggering pathways related to cell clustering. Overall, this report outlines an approach to contain invasive tumors that can complement adjuvant interventions for invasive GBM such as radiation and chemotherapy.
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http://dx.doi.org/10.1002/adhm.201801076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657526PMC
February 2019

In Vitro Transcribed mRNA Vaccines with Programmable Stimulation of Innate Immunity.

Bioconjug Chem 2018 09 13;29(9):3072-3083. Epub 2018 Aug 13.

Wallace H. Coulter Department of Biomedical Engineering , Georgia Institute of Technology and Emory University , Krone Engineering Biosystems Building, 950 Atlantic Drive , Atlanta , Georgia 30332 , United States.

In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manufactured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing translation of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immunostimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tethered IVT mRNA-TLR7 agonists leads to enhanced local immune responses, and to antigen-specific cell-mediated and humoral responses. We believe this system represents a potential platform compatible with any adjuvant of interest to enable specific programmable stimulation of immune responses.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00443DOI Listing
September 2018

Cerivastatin Nanoliposome as a Potential Disease Modifying Approach for the Treatment of Pulmonary Arterial Hypertension.

J Pharmacol Exp Ther 2018 07 25;366(1):66-74. Epub 2018 Apr 25.

Indiana Center for Biomedical Innovation, School of Medicine, Indiana University, Indianapolis, Indiana (Y.L., J.S.); Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and School of Medicine, Emory University, Atlanta, Georgia (S.B.P., R.V.B.); Department of Biomedical Engineering, Duke University, Durham, North Carolina (R.V.B.); and Department of Chemistry, Purdue University, West Lafayette, Indiana (Y.L., D.H.T.)

In this study we investigated nanoliposome as an approach to tailoring the pharmacology of cerivastatin as a disease-modifying drug for pulmonary arterial hypertension (PAH). Cerivastatin encapsulated liposomes with an average diameter of 98 ± 27 nm were generated by a thin film and freeze-thaw process. The nanoliposomes demonstrated sustained drug-release kinetics in vitro and inhibited proliferation of pulmonary artery (PA) smooth muscle cells with significantly less cellular cytotoxicity as compared with free cerivastatin. When delivered by inhalation to a rat model of monocrotaline-induced PAH, cerivastatin significantly reduced PA pressure from 55.13 ± 9.82 to 35.56 ± 6.59 mm Hg ( < 0.001) and diminished PA wall thickening. Echocardiography showed that cerivastatin significantly reduced right ventricle thickening (monocrotaline: 0.34 ± 0.02 cm vs. cerivastatin: 0.26 ± 0.02 cm; < 0.001) and increased PA acceleration time (monocrotaline: 13.98 ± 1.14 milliseconds vs. cerivastatin: 21.07 ± 2.80 milliseconds; < 0.001). Nanoliposomal cerivastatin was equally effective or slightly better than cerivastatin in reducing PA pressure (monocrotaline: 67.06 ± 13.64 mm Hg; cerivastatin: 46.31 ± 7.64 mm Hg vs. liposomal cerivastatin: 37.32 ± 9.50 mm Hg) and improving parameters of right ventricular function as measured by increasing PA acceleration time (monocrotaline: 24.68 ± 3.92 milliseconds; cerivastatin: 32.59 ± 6.10 milliseconds vs. liposomal cerivastatin: 34.96 ± 7.51 milliseconds). More importantly, the rate and magnitude of toxic cerivastatin metabolite lactone generation from the intratracheally administered nanoliposomes was significantly lower as compared with intravenously administered free cerivastatin. These studies show that nanoliposome encapsulation improved in vitro and in vivo pharmacologic and safety profile of cerivastatin and may represent a safer approach as a disease-modifying therapy for PAH.
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http://dx.doi.org/10.1124/jpet.118.247643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987999PMC
July 2018

Discovery of Lipidome Alterations Following Traumatic Brain Injury via High-Resolution Metabolomics.

J Proteome Res 2018 06 27;17(6):2131-2143. Epub 2018 Apr 27.

Wallace H Coulter Department of Biomedical Engineering , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.

Traumatic brain injury (TBI) can occur across wide segments of the population, presenting in a heterogeneous manner that makes diagnosis inconsistent and management challenging. Biomarkers offer the potential to objectively identify injury status, severity, and phenotype by measuring the relative concentrations of endogenous molecules in readily accessible biofluids. Through a data-driven, discovery approach, novel biomarker candidates for TBI were identified in the serum lipidome of adult male Sprague-Dawley rats in the first week following moderate controlled cortical impact (CCI). Serum samples were analyzed in positive and negative modes by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). A predictive panel for the classification of injured and uninjured sera samples, consisting of 26 dysregulated species belonging to a variety of lipid classes, was developed with a cross-validated accuracy of 85.3% using omniClassifier software to optimize feature selection. Polyunsaturated fatty acids (PUFAs) and PUFA-containing diacylglycerols were found to be upregulated in sera from injured rats, while changes in sphingolipids and other membrane phospholipids were also observed, many of which map to known secondary injury pathways. Overall, the identified biomarker panel offers viable molecular candidates representing lipids that may readily cross the blood-brain barrier (BBB) and aid in the understanding of TBI pathophysiology.
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http://dx.doi.org/10.1021/acs.jproteome.8b00068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341947PMC
June 2018

Correlation of mRNA Expression and Signal Variability in Chronic Intracortical Electrodes.

Front Bioeng Biotechnol 2018 27;6:26. Epub 2018 Mar 27.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC, United States.

Objective: The goal for this research was to identify molecular mechanisms that explain animal-to-animal variability in chronic intracortical recordings.

Approach: Microwire electrodes were implanted into Sprague Dawley rats at an acute (1 week) and a chronic (14 weeks) time point. Weekly recordings were conducted, and action potentials were evoked in the barrel cortex by deflecting the rat's whiskers. At 1 and 14 weeks, tissue was collected, and mRNA was extracted. mRNA expression was compared between 1 and 14 weeks using a high throughput multiplexed qRT-PCR. Pearson correlation coefficients were calculated between mRNA expression and signal-to-noise ratios at 14 weeks.

Main Results: At 14 weeks, a positive correlation between signal-to-noise ratio (SNR) and NeuN and GFAP mRNA expression was observed, indicating a relationship between recording strength and neuronal population, as well as reactive astrocyte activity. The inflammatory state around the electrode interface was evaluated using M1-like and M2-like markers. Expression for both M1-like and M2-like mRNA markers remained steady from 1 to 14 weeks. Anti-inflammatory markers, CD206 and CD163, however, demonstrated a significant positive correlation with SNR quality at 14 weeks. VE-cadherin, a marker for adherens junctions, and PDGFR-β, a marker for pericytes, both partial representatives of blood-brain barrier health, had a positive correlation with SNR at 14 weeks. Endothelial adhesion markers revealed a significant increase in expression at 14 weeks, while CD45, a pan-leukocyte marker, significantly decreased at 14 weeks. No significant correlation was found for either the endothelial adhesion or pan-leukocyte markers.

Significance: A positive correlation between anti-inflammatory and blood-brain barrier health mRNA markers with electrophysiological efficacy of implanted intracortical electrodes has been demonstrated. These data reveal potential mechanisms for further evaluation to determine potential target mechanisms to improve consistency of intracortical electrodes recordings and reduce animal-to-animal/implant-to-implant variability.
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http://dx.doi.org/10.3389/fbioe.2018.00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880884PMC
March 2018

Therapeutic efficacy of microtube-embedded chondroitinase ABC in a canine clinical model of spinal cord injury.

Brain 2018 04;141(4):1017-1027

1 College of Veterinary Medicine, Iowa State University, 1800 Christensen Drive, Ames IA 50011, USA.

See Moon and Bradbury (doi:10.1093/brain/awy067) for a scientific commentary on this article.Many hundreds of thousands of people around the world are living with the long-term consequences of spinal cord injury and they need effective new therapies. Laboratory research in experimental animals has identified a large number of potentially translatable interventions but transition to the clinic is not straightforward. Further evidence of efficacy in more clinically-relevant lesions is required to gain sufficient confidence to commence human clinical trials. Of the many therapeutic candidates currently available, intraspinally applied chondroitinase ABC has particularly well documented efficacy in experimental animals. In this study we measured the effects of this intervention in a double-blinded randomized controlled trial in a cohort of dogs with naturally-occurring severe chronic spinal cord injuries that model the condition in humans. First, we collected baseline data on a series of outcomes: forelimb-hindlimb coordination (the prespecified primary outcome measure), skin sensitivity along the back, somatosensory evoked and transcranial magnetic motor evoked potentials and cystometry in 60 dogs with thoracolumbar lesions. Dogs were then randomized 1:1 to receive intraspinal injections of heat-stabilized, lipid microtube-embedded chondroitinase ABC or sham injections consisting of needle puncture of the skin. Outcome data were measured at 1, 3 and 6 months after intervention; skin sensitivity was also measured 24 h after injection (or sham). Forelimb-hindlimb coordination was affected by neither time nor chondroitinase treatment alone but there was a significant interaction between these variables such that coordination between forelimb and hindlimb stepping improved during the 6-month follow-up period in the chondroitinase-treated animals by a mean of 23%, but did not change in controls. Three dogs (10%) in the chondroitinase group also recovered the ability to ambulate without assistance. Sensitivity of the dorsal skin increased at 24 h after intervention in both groups but subsequently decreased to normal levels. Cystometry identified a non-significant improvement of bladder compliance at 1 month in the chondroitinase-injected dogs but this did not persist. There were no overall differences between groups in detection of sensory evoked potentials. Our results strongly support a beneficial effect of intraspinal injection of chondroitinase ABC on spinal cord function in this highly clinically-relevant model of chronic severe spinal cord injury. There was no evidence of long-term adverse effects associated with this intervention. We therefore conclude that this study provides strong evidence in support of initiation of clinical trials of chondroitinase ABC in humans with chronic spinal cord injury.
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http://dx.doi.org/10.1093/brain/awy007DOI Listing
April 2018

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels.

Biomaterials 2017 Oct 10;142:52-61. Epub 2017 Jul 10.

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA. Electronic address:

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CCX3CR1 non-classical monocytes and CD206 M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.
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http://dx.doi.org/10.1016/j.biomaterials.2017.07.013DOI Listing
October 2017

Engineering challenges for brain tumor immunotherapy.

Adv Drug Deliv Rev 2017 05 15;114:19-32. Epub 2017 Jun 15.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 101 Science Drive, Durham, NC 27708-0271, USA. Electronic address:

Malignant brain tumors represent one of the most devastating forms of cancer with abject survival rates that have not changed in the past 60years. This is partly because the brain is a critical organ, and poses unique anatomical, physiological, and immunological barriers. The unique interplay of these barriers also provides an opportunity for creative engineering solutions. Cancer immunotherapy, a means of harnessing the host immune system for anti-tumor efficacy, is becoming a standard approach for treating many cancers. However, its use in brain tumors is not widespread. This review discusses the current approaches, and hurdles to these approaches in treating brain tumors, with a focus on immunotherapies. We identify critical barriers to immunoengineering brain tumor therapies and discuss possible solutions to these challenges.
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http://dx.doi.org/10.1016/j.addr.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870902PMC
May 2017

Immunoengineering nerve repair.

Proc Natl Acad Sci U S A 2017 06 13;114(26):E5077-E5084. Epub 2017 Jun 13.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, NC 27708;

Injuries to the peripheral nervous system are major sources of disability and often result in painful neuropathies or the impairment of muscle movement and/or normal sensations. For gaps smaller than 10 mm in rodents, nearly normal functional recovery can be achieved; for longer gaps, however, there are challenges that have remained insurmountable. The current clinical gold standard used to bridge long, nonhealing nerve gaps, the autologous nerve graft (autograft), has several drawbacks. Despite best efforts, engineering an alternative "nerve bridge" for peripheral nerve repair remains elusive; hence, there is a compelling need to design new approaches that match or exceed the performance of autografts across critically sized nerve gaps. Here an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.
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http://dx.doi.org/10.1073/pnas.1705757114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495274PMC
June 2017

Bacterial Carriers for Glioblastoma Therapy.

Mol Ther Oncolytics 2017 Mar 14;4:1-17. Epub 2016 Dec 14.

Department of Biomedical Engineering, Pratt School of Engineering, Duke University, 101 Science Drive, Durham, NC 27708-0271, USA.

Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these barriers, bacterial carriers that are actively motile and programmed to migrate and localize to tumor zones were designed. These carriers can induce apoptosis via hypoxia-controlled expression of a tumor suppressor protein p53 and a pro-apoptotic drug, Azurin. In a xenograft model of human glioblastoma in rats, bacterial carrier therapy conferred a significant survival benefit with 19% overall long-term survival of >100 days in treated animals relative to a median survival of 26 days in control untreated animals. Histological and proteomic analyses were performed to elucidate the safety and efficacy of these carriers, showing an absence of systemic toxicity and a restored neural environment in treated responders. In the treated non-responders, proteomic analysis revealed competing mechanisms of pro-apoptotic and drug-resistant activity. This bacterial carrier opens a versatile avenue to overcome diffusion barriers in glioblastoma by virtue of its active motility in extracellular space and can lead to tailored therapies via tumor-specific expression of tumoricidal proteins.
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http://dx.doi.org/10.1016/j.omto.2016.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363759PMC
March 2017

Chondroitin Sulfate Glycosaminoglycan Matrices Promote Neural Stem Cell Maintenance and Neuroprotection Post-Traumatic Brain Injury.

ACS Biomater Sci Eng 2017 Mar 13;3(3):420-430. Epub 2017 Feb 13.

Regenerative Bioscience Center, The University of Georgia, 425 River Road, ADS Complex, Athens, Georgia 30602, United States.

There are currently no effective treatments for moderate-to-severe traumatic brain injuries (TBIs). The paracrine functions of undifferentiated neural stem cells (NSCs) are believed to play a significant role in stimulating the repair and regeneration of injured brain tissue. We therefore hypothesized that fibroblast growth factor (FGF2) enriching chondroitin sulfate glycosaminoglycan (CS-GAG) matrices can maintain the undifferentiated state of neural stem cells (NSCs) and facilitate brain tissue repair subacutely post-TBI. Rats subjected to a controlled cortical impactor (CCI) induced TBI were intraparenchymally injected with CS-GAG matrices alone or with CS-GAG matrices containing PKH26GL labeled allogeneic NSCs. Nissl staining of brain tissue 4 weeks post-TBI demonstrated the significantly enhanced ( < 0.05) tissue protection in CS-GAG treated animals when compared to TBI only control, and NSC only treated animals. CS-GAG-NSC treated animals demonstrated significantly enhanced ( < 0.05) FGF2 retention, and maintenance of PKH26GL labeled NSCs as indicated by enhanced Sox1+ and Ki67+ cell presence over other differentiated cell types. Lastly, all treatment groups and sham controls exhibited a significantly ( < 0.05) attenuated GFAP+ reactive astrocyte presence in the lesion site when compared to TBI only controls.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937277PMC
March 2017

Kilohertz frequency nerve block enhances anti-inflammatory effects of vagus nerve stimulation.

Sci Rep 2017 01 5;7:39810. Epub 2017 Jan 5.

Georgia Institute of Technology, Department of Biomedical Engineering, Atlanta, GA, 30332 USA.

Efferent activation of the cervical vagus nerve (cVN) dampens systemic inflammatory processes, potentially modulating a wide-range of inflammatory pathological conditions. In contrast, afferent cVN activation amplifies systemic inflammatory processes, leading to activation of the hypothalamic-pituitary-adrenal (HPA) axis, the sympathetic nervous system through the greater splanchnic nerve (GSN), and elevation of pro-inflammatory cytokines. Ideally, to clinically implement anti-inflammatory therapy via cervical vagus nerve stimulation (cVNS) one should selectively activate the efferent pathway. Unfortunately, current implementations, in animal and clinical investigations, activate both afferent and efferent pathways. We paired cVNS with kilohertz electrical stimulation (KES) nerve block to preferentially activate efferent pathways while blocking afferent pathways. Selective efferent cVNS enhanced the anti-inflammatory effects of cVNS. Our results demonstrate that: (i) afferent, but not efferent, cVNS synchronously activates the GSN in a dose-dependent manner; (ii) efferent cVNS enabled by complete afferent KES nerve block enhances the anti-inflammatory benefits of cVNS; and (iii) incomplete afferent KES nerve block exacerbates systemic inflammation. Overall, these data demonstrate the utility of paired efferent cVNS and afferent KES nerve block for achieving selective efferent cVNS, specifically as it relates to neuromodulation of systemic inflammation.
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http://dx.doi.org/10.1038/srep39810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215548PMC
January 2017

Strategies for modulating innate immune activation and protein production of in vitro transcribed mRNAs.

J Mater Chem B 2016 Mar 13;4(9):1619-1632. Epub 2015 Oct 13.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, USA.

Synthetic mRNA has recently shown great potential as a tool for genetic introduction of proteins. Its utility as a gene carrier has been demonstrated in several studies for both the introduction of therapeutic proteins and subunit vaccines. At one point, synthetic mRNA was believed to be too immunogenic and labile for pharmaceutical purposes. However, the development of several strategies have enabled mRNA technology to overcome these challenges, including incorporation of modified nucleotides, codon optimization of the coding region, incorporation of untranslated regions into the mRNA, and the use of delivery vehicles. While these approaches have been shown to enhance performance of some mRNA constructs, gene-to-gene variation and low efficiency of mRNA protein production are still significant hurdles. Further mechanistic understanding of how these strategies affect protein production and innate immune activation is needed for the widespread adoption for both therapeutic and vaccine applications. This review highlights key studies involved in the development of strategies employed to increase protein expression and control the immunogenicity of synthetic mRNA. Areas in the literature where improved understanding is needed will also be discussed.
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http://dx.doi.org/10.1039/c5tb01753jDOI Listing
March 2016

Response reliability observed with voltage-sensitive dye imaging of cortical layer 2/3: the probability of activation hypothesis.

J Neurophysiol 2016 06 10;115(5):2456-69. Epub 2016 Feb 10.

Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, Georgia

A central assertion in the study of neural processing is that our perception of the environment directly reflects the activity of our sensory neurons. This assertion reinforces the intuition that the strength of a sensory input directly modulates the amount of neural activity observed in response to that sensory feature: an increase in the strength of the input yields a graded increase in the amount of neural activity. However, cortical activity across a range of sensory pathways can be sparse, with individual neurons having remarkably low firing rates, often exhibiting suprathreshold activity on only a fraction of experimental trials. To compensate for this observed apparent unreliability, it is assumed that instead the local population of neurons, although not explicitly measured, does reliably represent the strength of the sensory input. This assumption, however, is largely untested. In this study, using wide-field voltage-sensitive dye (VSD) imaging of the somatosensory cortex in the anesthetized rat, we show that whisker deflection velocity, or stimulus strength, is not encoded by the magnitude of the population response at the level of cortex. Instead, modulation of whisker deflection velocity affects the likelihood of the cortical response, impacting the magnitude, rate of change, and spatial extent of the cortical response. An ideal observer analysis of the cortical response points to a probabilistic code based on repeated sampling across cortical columns and/or time, which we refer to as the probability of activation hypothesis. This hypothesis motivates a range of testable predictions for both future electrophysiological and future behavioral studies.
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http://dx.doi.org/10.1152/jn.00547.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922466PMC
June 2016

Implantable electronics: A sensor web for neurons.

Nat Mater 2015 Dec;14(12):1190-1

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, UA Whitaker Building, 313 Ferst Drive, Atlanta, Georgia 30332, USA.

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http://dx.doi.org/10.1038/nmat4454DOI Listing
December 2015

Chondroitin Sulfate Glycosaminoglycan Hydrogels Create Endogenous Niches for Neural Stem Cells.

Bioconjug Chem 2015 Dec 20;26(12):2336-49. Epub 2015 Oct 20.

Regenerative Bioscience Center, ADS Complex, The University of Georgia , 425 River Road, Athens, Georgia 30602, United States.

Neural stem cells (NSCs) possess great potential for neural tissue repair after traumatic injuries to the central nervous system (CNS). However, poor survival and self-renewal of NSCs after injury severely limits its therapeutic potential. Sulfated chondroitin sulfate glycosaminoglycans (CS-GAGs) linked to CS proteoglycans (CSPGs) in the brain extracellular matrix (ECM) have the ability to bind and potentiate trophic factor efficacy, and promote NSC self-renewal in vivo. In this study, we investigated the potential of CS-GAG hydrogels composed of monosulfated CS-4 (CS-A), CS-6 (CS-C), and disulfated CS-4,6 (CS-E) CS-GAGs as NSC carriers, and their ability to create endogenous niches by enriching specific trophic factors to support NSC self-renewal. We demonstrate that CS-GAG hydrogel scaffolds showed minimal swelling and degradation over a period of 15 days in vitro, absorbing only 6.5 ± 0.019% of their initial weight, and showing no significant loss of mass during this period. Trophic factors FGF-2, BDNF, and IL10 bound with high affinity to CS-GAGs, and were significantly (p < 0.05) enriched in CS-GAG hydrogels when compared to unsulfated hyaluronic acid (HA) hydrogels. Dissociated rat subventricular zone (SVZ) NSCs when encapsulated in CS-GAG hydrogels demonstrated ∼88.5 ± 6.1% cell viability in vitro. Finally, rat neurospheres in CS-GAG hydrogels conditioned with the mitogen FGF-2 demonstrated significantly (p < 0.05) higher self-renewal when compared to neurospheres cultured in unconditioned hydrogels. Taken together, these findings demonstrate the ability of CS-GAG based hydrogels to regulate NSC self-renewal, and facilitate growth factor enrichment locally.
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http://dx.doi.org/10.1021/acs.bioconjchem.5b00397DOI Listing
December 2015

A regenerative microchannel device for recording multiple single-unit action potentials in awake, ambulatory animals.

Eur J Neurosci 2016 Feb 28;43(3):474-85. Epub 2015 Oct 28.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, 313 Ferst Drive, Atlanta, GA, 30332, USA.

Despite significant advances in robotics, commercially advanced prosthetics provide only a small fraction of the functionality of the amputated limb that they are meant to replace. Peripheral nerve interfacing could provide a rich controlling link between the body and these advanced prosthetics in order to increase their overall utility. Here, we report on the development of a fully integrated regenerative microchannel interface with 30 microelectrodes and signal extraction capabilities enabling evaluation in an awake and ambulatory rat animal model. In vitro functional testing validated the capability of the microelectrodes to record neural signals similar in size and nature to those that occur in vivo. In vitro dorsal root ganglia cultures revealed striking cytocompatibility of the microchannel interface. Finally, in vivo, the microchannel interface was successfully used to record a multitude of single-unit action potentials through 63% of the integrated microelectrodes at the early time point of 3 weeks. This marks a significant advance in microchannel interfacing, demonstrating the capability of microchannels to be used for peripheral nerve interfacing.
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http://dx.doi.org/10.1111/ejn.13080DOI Listing
February 2016

Enhanced therapeutic neovascularization by CD31-expressing cells and embryonic stem cell-derived endothelial cells engineered with chitosan hydrogel containing VEGF-releasing microtubes.

Biomaterials 2015 Sep 11;63:158-67. Epub 2015 Jun 11.

Department of Medicine, Division of Cardiology, Emory University School of Medicine, 101 Woodruff Circle, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, 313 Ferst Drive, Atlanta, GA 30332, USA; Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul 120-752, South Korea. Electronic address:

Various stem cells and their progeny have been used therapeutically for vascular regeneration. One of the major hurdles for cell-based therapy is low cell retention in vivo, and to improve cell survival several biomaterials have been used to encapsulate cells before transplantation. Vascular regeneration involves new blood vessel formation which consists of two processes, vasculogenesis and angiogenesis. While embryonic stem cell (ESC)-derived endothelial cells (ESC-ECs) have clearer vasculogenic potency, adult cells exert their effects mainly through paracrine angiogenic activities. While these two cells have seemingly complementary advantages, there have not been any studies to date combining these two cell types for vascular regeneration. We have developed a novel chitosan-based hydrogel construct that encapsulates both CD31-expressing BM-mononuclear cells (BM-CD31(+) cells) and ESC-ECs, and is loaded with VEGF-releasing microtubes. This cell construct showed high cell survival and minimal cytotoxicity in vitro. When implanted into a mouse model of hindlimb ischemia, it induced robust cell retention, neovascularization through vasculogenesis and angiogenesis, and efficiently induced recovery of blood flow in ischemic hindlimbs. This chitosan-based hydrogel encapsulating mixed adult and embryonic cell derivatives and containing VEGF can serve as a novel platform for treating various cardiovascular diseases.
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http://dx.doi.org/10.1016/j.biomaterials.2015.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489430PMC
September 2015

Evans blue nanocarriers visually demarcate margins of invasive gliomas.

Drug Deliv Transl Res 2015 Apr;5(2):116-24

Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, Atlanta, GA, USA.

Aggressive surgical resection is the primary therapy for glioma. However, aggressive resection may compromise functional healthy brain tissue. Currently, there are no objective cues for surgeons to distinguish healthy tissue from tumor and determine tumor borders; surgeons skillfully rely on subjective means such as tactile feedback. This often results in incomplete resection and recurrence. The objective of the present study was to design, develop, and evaluate, in vitro and in vivo, a nanoencapsulated visible dye for intraoperative, visual delineation of tumor margins in an invasive tumor model. Liposomal nanocarriers containing Evans blue dye (nano-EB) were developed, characterized, and tested for safety in vitro and in vivo. 3RT1RT2A glioma cells were implanted into brains of Fischer 344 rats. Nano-EB or EB solution was injected via tail vein into tumor-bearing animals. To assess tumor staining, tissue samples were analyzed visibly and using fluorescence microscopy. Area, perimeter ratios, and Manders overlap coefficients were calculated to quantify extent of staining. Nano-EB clearly marked tumor margins in the invasive tumor model. Area ratio of nano-EB staining to tumor was 0.89 ± 0.05, perimeter ratio was 0.94 ± 0.04, Manders R was 0.51 ± 0.08, and M1 was 0.97 ± 0.06. Microscopic tumor border inspection under high magnification verified that nano-EB did not stain healthy tissue. Nano-EB clearly aids in distinguishing tumor tissue from healthy tissue in an invasive tumor model, while injection of unencapsulated EB results in false identification of healthy tissue as tumor due to diffusion of dye from the tumor into healthy tissue.
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http://dx.doi.org/10.1007/s13346-013-0139-xDOI Listing
April 2015

Noninvasive imaging of peripheral nerves.

Cells Tissues Organs 2014 4;200(1):69-77. Epub 2015 Mar 4.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Ga., USA.

Recent developments in the field of peripheral nerve imaging extend the capabilities of imaging modalities to assist in the diagnosis and treatment of patients with peripheral nerve maladies. Methods such as magnetic resonance imaging (MRI) and its derivative diffusion tensor imaging (DTI), ultrasound (US) and positron emission tomography (PET) are capable of assessing nerve structure and function following injury and relating the state of the nerve to electrophysiological and histological analysis. Of the imaging methods surveyed here, each offered unique and interesting advantages related to the field. MRI offered the opportunity to visualize immune activity on the injured nerve throughout the course of the regeneration process, and DTI offered numerical characterization of the injury and the ability to develop statistical bases for diagnosing injury. US extends imaging to the treatment phase by enabling more precise analgesic applications following surgery, and PET represents a novel method of assessing nerve injury through analysis of relative metabolism rates in injured and healthy tissue. Exciting new possibilities to enhance and extend the abilities of imaging methods are also discussed, including innovative contrast agents, some of which enable multimodal imaging approaches and present opportunities for treatment application.
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http://dx.doi.org/10.1159/000369451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494672PMC
November 2015

Protease-degradable PEG-maleimide coating with on-demand release of IL-1Ra to improve tissue response to neural electrodes.

Biomaterials 2015 Mar 12;44:55-70. Epub 2015 Jan 12.

Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA; Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. Electronic address:

Neural electrodes are an important part of brain-machine interface devices that can restore functionality to patients with sensory and movement disorders. Chronically implanted neural electrodes induce an unfavorable tissue response which includes inflammation, scar formation, and neuronal cell death, eventually causing loss of electrode function. We developed a poly(ethylene glycol) hydrogel coating for neural electrodes with non-fouling characteristics, incorporated an anti-inflammatory agent, and engineered a stimulus-responsive degradable portion for on-demand release of the anti-inflammatory agent in response to inflammatory stimuli. This coating reduces in vitro glial cell adhesion, cell spreading, and cytokine release compared to uncoated controls. We also analyzed the in vivo tissue response using immunohistochemistry and microarray qRT-PCR. Although no differences were observed among coated and uncoated electrodes for inflammatory cell markers, lower IgG penetration into the tissue around PEG+IL-1Ra coated electrodes indicates an improvement in blood-brain barrier integrity. Gene expression analysis showed higher expression of IL-6 and MMP-2 around PEG+IL-1Ra samples, as well as an increase in CNTF expression, an important marker for neuronal survival. Importantly, increased neuronal survival around coated electrodes compared to uncoated controls was observed. Collectively, these results indicate promising findings for an engineered coating to increase neuronal survival and improve tissue response around implanted neural electrodes.
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http://dx.doi.org/10.1016/j.biomaterials.2014.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4307944PMC
March 2015

Nanocarrier-mediated inhibition of macrophage migration inhibitory factor attenuates secondary injury after spinal cord injury.

ACS Nano 2015 Feb 28;9(2):1492-505. Epub 2015 Jan 28.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine , Atlanta, Georgia 30332, United States.

Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity, improved wound healing, and an increase in levels of arginase and other transcripts indicative of a resolution phase of wound healing. This study demonstrates the potential of MIF inhibition in SCI and the utility of nanocarrier-mediated drug delivery selectively to the injured cord.
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http://dx.doi.org/10.1021/nn505980zDOI Listing
February 2015

Extracellular matrix-based intracortical microelectrodes: Toward a microfabricated neural interface based on natural materials.

Microsyst Nanoeng 2015 29;1(1). Epub 2015 Jun 29.

Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332, USA.

Extracellular matrix (ECM)-based implantable neural electrodes (NEs) were achieved using a microfabrication strategy on natural-substrate-based organic materials. The ECM-based design minimized the introduction of non-natural products into the brain. Further, it rendered the implants sufficiently rigid for penetration into the target brain region and allowed them subsequently to soften to match the elastic modulus of brain tissue upon exposure to physiological conditions, thereby reducing inflammatory strain fields in the tissue. Preliminary studies suggested that ECM-NEs produce a reduced inflammatory response compared with inorganic rigid and flexible approaches. intracortical recordings from the rat motor cortex illustrate one mode of use for these ECM-NEs.
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http://dx.doi.org/10.1038/micronano.2015.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258041PMC
June 2015

Microchannel-based regenerative scaffold for chronic peripheral nerve interfacing in amputees.

Biomaterials 2015 Feb 9;41:151-65. Epub 2014 Dec 9.

Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA. Electronic address:

Neurally controlled prosthetics that cosmetically and functionally mimic amputated limbs remain a clinical need because state of the art neural prosthetics only provide a fraction of a natural limb's functionality. Here, we report on the fabrication and capability of polydimethylsiloxane (PDMS) and epoxy-based SU-8 photoresist microchannel scaffolds to serve as viable constructs for peripheral nerve interfacing through in vitro and in vivo studies in a sciatic nerve amputee model where the nerve lacks distal reinnervation targets. These studies showed microchannels with 100 μm × 100 μm cross-sectional areas support and direct the regeneration/migration of axons, Schwann cells, and fibroblasts through the microchannels with space available for future maturation of the axons. Investigation of the nerve in the distal segment, past the scaffold, showed a high degree of organization, adoption of the microchannel architecture forming 'microchannel fascicles', reformation of endoneurial tubes and axon myelination, and a lack of aberrant and unorganized growth that might be characteristic of neuroma formation. Separate chronic terminal in vivo electrophysiology studies utilizing the microchannel scaffolds with permanently integrated microwire electrodes were conducted to evaluate interfacing capabilities. In all devices a variety of spontaneous, sensory evoked and electrically evoked single and multi-unit action potentials were recorded after five months of implantation. Together, these findings suggest that microchannel scaffolds are well suited for chronic implantation and peripheral nerve interfacing to promote organized nerve regeneration that lends itself well to stable interfaces. Thus this study establishes the basis for the advanced fabrication of large-electrode count, wireless microchannel devices that are an important step towards highly functional, bi-directional peripheral nerve interfaces.
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http://dx.doi.org/10.1016/j.biomaterials.2014.11.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324612PMC
February 2015