Publications by authors named "Lohitash Karumbaiah"

33 Publications

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abe0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935369PMC
March 2021

Neurostimulation and Reach-to-Grasp Function Recovery Following Acquired Brain Injury: Insight From Pre-clinical Rodent Models and Human Applications.

Front Neurol 2020 21;11:835. Epub 2020 Jul 21.

Regenerative Bioscience Center, University of Georgia, Athens, GA, United States.

Reach-to-grasp is an evolutionarily conserved motor function that is adversely impacted following stroke and traumatic brain injury (TBI). Non-invasive brain stimulation (NIBS) methods, such as transcranial magnetic stimulation and transcranial direct current stimulation, are promising tools that could enhance functional recovery of reach-to-grasp post-brain injury. Though the rodent literature provides a causal understanding of post-injury recovery mechanisms, it has had a limited impact on NIBS protocols in human research. The high degree of homology in reach-to-grasp circuitry between humans and rodents further implies that the application of NIBS to brain injury could be better informed by findings from pre-clinical rodent models and neurorehabilitation research. Here, we provide an overview of the advantages and limitations of using rodent models to advance our current understanding of human reach-to-grasp function, cortical circuitry, and reorganization. We propose that a cross-species comparison of reach-to-grasp recovery could provide a mechanistic framework for clinically efficacious NIBS treatments that could elicit better functional outcomes for patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fneur.2020.00835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7396659PMC
July 2020

Label-free ferrohydrodynamic separation of exosome-like nanoparticles.

Lab Chip 2020 08;20(17):3187-3201

School of Electrical and Computer Engineering, The University of Georgia, Athens, GA 30602, USA.

Isolation of exosomes from biological samples provides a minimally-invasive alternative for basic understanding, diagnosis, and prognosis of metastatic cancers. The biology and clinical values of exosomes are under intensive investigation, yet most studies are limited by technical challenges in recovering these exosomes with heterogeneous sizes and cargos from biological samples. We report a novel method based on "particle ferrohydrodynamics" and its associated microfluidic device, termed as the FerroChip, which can separate exosome-like nanoparticles from microliters of cell culture media and human serum in a label-free, continuous-flow and size-dependent manner, and achieves a high recovery rate (94.3%) and a high purity (87.9%). Separated exosome-like nanoparticles had diameters, morphology, and protein expressions that were consistent with other reports. This method, upon further molecular characterization, could potentially facilitate basic understanding of exosomes and its clinical application in blood liquid biopsy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/d0lc00609bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7493820PMC
August 2020

Cortical Transplantation of Brain-Mimetic Glycosaminoglycan Scaffolds and Neural Progenitor Cells Promotes Vascular Regeneration and Functional Recovery after Ischemic Stroke in Mice.

Adv Healthc Mater 2020 03 24;9(5):e1900285. Epub 2020 Jan 24.

Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, 30322, USA.

Stroke causes significant mortality and morbidity. Currently, there are no treatments which can regenerate brain tissue lost to infarction. Neural progenitor cells (NPCs) are at the forefront of preclinical studies for regenerative stroke therapies. NPCs can differentiate into and replace neurons and promote endogenous recovery mechanisms such as angiogenesis via trophic factor production and release. The stroke core is hypothetically the ideal location for replacement of neural tissue since it is in situ and develops into a potential space where injections may be targeted with minimal compression of healthy peri-infarct tissue. However, the compromised perfusion and tissue degradation following ischemia create an inhospitable environment resistant to cellular therapy. Overcoming these limitations is critical to advancing cellular therapy. In this work, the therapeutic potential of mouse-induced pluripotent stem cell derived NPCs is tested encapsulated in a basic fibroblast growth factor (bFGF) binding chondroitin sulfate-A (CS-A) hydrogel transplanted into the infarct core in a mouse sensorimotor cortex mini-stroke model. It is shown that CS-A encapsulation significantly improves vascular remodeling, cortical blood flow, and sensorimotor behavioral outcomes after stroke. It is found these improvements are negated by blocking bFGF, suggesting that the sustained trophic signaling endowed by the CS-A hydrogel combined with NPC transplantation can promote tissue repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201900285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7358896PMC
March 2020

Extracellular Vesicles Mediate Neuroprotection and Functional Recovery after Traumatic Brain Injury.

J Neurotrauma 2020 06 10;37(11):1358-1369. Epub 2020 Feb 10.

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

The lack of effective therapies for moderate-to-severe traumatic brain injuries (TBIs) leaves patients with lifelong disabilities. Neural stem cells (NSCs) have demonstrated great promise for neural repair and regeneration. However, direct evidence to support their use as a cell replacement therapy for neural injuries is currently lacking. We hypothesized that NSC-derived extracellular vesicles (NSC EVs) mediate repair indirectly after TBI by enhancing neuroprotection and therapeutic efficacy of endogenous NSCs. We evaluated the short-term effects of acute intravenous injections of NSC EVs immediately following a rat TBI. Male NSC EV-treated rats demonstrated significantly reduced lesion sizes, enhanced presence of endogenous NSCs, and attenuated motor function impairments 4 weeks post-TBI, when compared with vehicle- and TBI-only male controls. Although statistically not significant, we observed a therapeutic effect of NSC EVs on brain lesion volume, nestin expression, and behavioral recovery in female subjects. Our study demonstrates the neuroprotective and functional benefits of NSC EVs for treating TBI and points to gender-dependent effects on treatment outcomes, which requires further investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/neu.2019.6443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249471PMC
June 2020

Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

FASEB J 2019 11 9;33(11):11973-11992. Epub 2019 Aug 9.

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

Invasive spread of glioblastoma (GBM) is linked to changes in chondroitin sulfate (CS) proteoglycan (CSPG)-associated sulfated glycosaminoglycans (GAGs) that are selectively up-regulated in the tumor microenvironment (TME). We hypothesized that inhibiting CS-GAG signaling in the TME would stem GBM invasion. Rat F98 GBM cells demonstrated enhanced preferential cell invasion into oversulfated 3-dimensional composite of CS-A and CS-E [4- and 4,6-sulfated CS-GAG (COMP)] matrices compared with monosulfated (4-sulfated) and unsulfated hyaluronic acid matrices in microfluidics-based choice assays, which is likely influenced by differential GAG receptor binding specificities. Both F98 and human patient-derived glioma stem cells (GSCs) demonstrated a high degree of colocalization of the GSC marker CD133 and CSPGs. The small molecule sulfated GAG antagonist bis-2-methyl-4-amino-quinolyl-6-carbamide (surfen) reduced invasion and focal adhesions in F98 cells encapsulated in COMP matrices and blocked CD133 and antichondroitin sulfate antibody (CS-56) detection of respective antigens in F98 cells and human GSCs. Surfen-treated F98 cells down-regulated CSPG-binding receptor transcripts and protein, as well as total and activated ERK and protein kinase B. Lastly, rats induced with frontal lobe tumors and treated with a single intratumoral dose of surfen demonstrated reduced tumor burden and spread compared with untreated controls. These results present a first demonstration of surfen as an inhibitor of sulfated GAG signaling to stem GBM invasion.-Logun, M. T., Wynens, K. E., Simchick, G., Zhao, W., Mao, L., Zhao, Q., Mukherjee, S., Brat, D. J., Karumbaiah, L. Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201802610RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902699PMC
November 2019

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells.

ACS Chem Biol 2019 09 21;14(9):1921-1929. Epub 2019 Aug 21.

Complex Carbohydrate Research Center , University of Georgia , 315 Riverbend Road , Athens , Georgia 30602 , United States.

Heparin and heparan sulfate (HS) are attractive components for constructing biomaterials due to their ability to recruit and regulate the activity of growth factors. The structural and functional heterogeneity of naturally derived heparin and HS is, however, an impediment for the preparation of biomaterials for regenerative medicine. To address this problem, we have prepared hydrogels modified by well-defined synthetic HS-derived disaccharides. Human induced pluripotent cell-derived neural stem cells (HIP-NSCs) encapsulated in a polyethylene glycol-based hydrogel modified by a pendent HS disaccharide that is a known ligand for fibroblast growth factor-2 (FGF-2) exhibited a significant increase in proliferation and self-renewal. This observation is important because evidence is emerging that undifferentiated stems cells can yield significant therapeutic benefits via their paracrine signaling mechanisms. Our data indicate that the HS disaccharide protects FGF-2, which has a very short biological half-live, from degradation. It is anticipated that, by careful selection of a synthetic HS oligosaccharide, it will be possible to control retention and release of specific growth factor, which in turn will provide control over cell fate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.9b00401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272102PMC
September 2019

Five-dimensional two-photon volumetric microscopy of in-vivo dynamic activities using liquid lens remote focusing.

Biomed Opt Express 2019 Jul 26;10(7):3591-3604. Epub 2019 Jun 26.

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

Multi-photon scanning microscopy provides a robust tool for optical sectioning, which can be used to capture fast biological events such as blood flow, mitochondrial activity, and neuronal action potentials. For many studies, it is important to visualize several different focal planes at a rate akin to the biological event frequency. Typically, a microscope is equipped with mechanical elements to move either the sample or the objective lens to capture volumetric information, but these strategies are limited due to their slow speeds or inertial artifacts. To overcome this problem, remote focusing methods have been developed to shift the focal plane axially without physical movement of the sample or the microscope. Among these methods is liquid lens technology, which adjusts the focus of the lens by changing the wettability of the liquid and hence its curvature. Liquid lenses are inexpensive active optical elements that have the potential for fast multi-photon volumetric imaging, hence a promising and accessible approach for the study of biological systems with complex dynamics. Although remote focusing using liquid lens technology can be used for volumetric point scanning multi-photon microscopy, optical aberrations and the effects of high energy laser pulses have been concerns in its implementation. In this paper, we characterize a liquid lens and validate its use in relevant biological applications. We measured optical aberrations that are caused by the liquid lens, and calculated its response time, defocus hysteresis, and thermal response to a pulsed laser. We applied this method of remote focusing for imaging and measurement of multiple in-vivo specimens, including mesenchymal stem cell dynamics, mouse tibialis anterior muscle mitochondrial electrical potential fluctuations, and mouse brain neural activity. Our system produces 5 dimensional (x,y,z,λ,t) data sets at the speed of 4.2 volumes per second over volumes as large as 160 x 160 x 35 µm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1364/BOE.10.003591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640832PMC
July 2019

Expanding Hydrophobically Modified Chitosan Foam for Internal Surgical Hemostasis: Safety Evaluation in a Murine Model.

J Surg Res 2019 07 16;239:269-277. Epub 2019 Mar 16.

Regenerative Bioscience Center, University of Georgia, Athens, Georgia. Electronic address:

Background: A novel injectable expanding foam based on hydrophobically modified chitosan (HM-CS) was developed to improve hemostasis during surgeries. HM-CS is an amphiphilic derivative of the natural biopolymer chitosan (CS); HM-CS has been shown to improve the natural hemostatic characteristics of CS, but its internal safety has not been systematically evaluated. The goal of this study was to compare the long-term in vivo safety of HM-CS relative to a commonly used fibrin sealant (FS), TISSEEL (Baxter).

Methods: Sixty-four Sprague-Dawley rats (275-325 g obtained from Charles River Laboratories) were randomly assigned to control (n = 16) or experimental (n = 48) groups. Samples of the test materials (HM-CS [n = 16], CS [n = 16], and FS [n = 16]) applied to a nonlethal liver excision (0.4 ± 0.3 g of the medial lobe) in rats were left inside the abdomen to degrade. Animals were observed daily for signs of morbidity and mortality. Surviving animals were sacrificed at 1 and 6 wk; the explanted injury sites were microscopically assessed.

Results: All animals (64/64) survived both the 1- and 6-wk time points without signs of morbidity. Histological examination showed a comparable pattern of degradation for the various test materials. FS remnants and significant adhesions to neighboring tissues were observed at 6 wk. Residual CS and HM-CS were observed at the 6 wk with fatty deposits at the site of injury. Minimal adhesions were observed for CS and HM-CS.

Conclusions: The internal safety observed in the HM-CS test group after abdominal implantation indicates that injectable HM-CS expanding foam may be an appropriate internal use hemostatic candidate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jss.2019.01.060DOI Listing
July 2019

Chondroitin Sulfate Glycosaminoglycan Scaffolds for Cell and Recombinant Protein-Based Bone Regeneration.

Stem Cells Transl Med 2019 06 21;8(6):575-585. Epub 2019 Jan 21.

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

Bone morphogenetic protein 2 (BMP-2)-loaded collagen sponges remain the clinical standard for treatment of large bone defects when there is insufficient autograft, despite associated complications. Recent efforts to negate comorbidities have included biomaterials and gene therapy approaches to extend the duration of BMP-2 release and activity. In this study, we compared the collagen sponge clinical standard to chondroitin sulfate glycosaminoglycan (CS-GAG) scaffolds as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) and rhBMP-2 expression via human BMP-2 gene inserted into mesenchymal stem cells (BMP-2 MSC). We demonstrated extended release of rhBMP-2 from CS-GAG scaffolds compared to their collagen sponge counterparts, and further extended release from CS-GAG gels seeded with BMP-2 MSC. When used to treat a challenging critically sized femoral defect model in rats, both rhBMP-2 and BMP-2 MSC in CS-GAG induced comparable bone formation to the rhBMP-2 in collagen sponge, as measured by bone volume, strength, and stiffness. We conclude that CS-GAG scaffolds are a promising delivery vehicle for controlling the release of rhBMP-2 and to mediate the repair of critically sized segmental bone defects. Stem Cells Translational Medicine 2019;8:575-585.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/sctm.18-0141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525555PMC
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201801076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657526PMC
February 2019

Chronic Electrical Stimulation Promotes the Excitability and Plasticity of ESC-derived Neurons following Glutamate-induced Inhibition In vitro.

Sci Rep 2018 Jul 19;8(1):10957. Epub 2018 Jul 19.

Regenerative Bioscience Center, ADS Complex, University of Georgia, Athens, Georgia.

Functional electrical stimulation (FES) is rapidly gaining traction as a therapeutic tool for mediating the repair and recovery of the injured central nervous system (CNS). However, the underlying mechanisms and impact of these stimulation paradigms at a molecular, cellular and network level remain largely unknown. In this study, we used embryonic stem cell (ESC)-derived neuron and glial co-cultures to investigate network maturation following acute administration of L-glutamate, which is a known mediator of excitotoxicity following CNS injury. We then modulated network maturation using chronic low frequency stimulation (LFS) and direct current stimulation (DCS) protocols. We demonstrated that L-glutamate impaired the rate of maturation of ESC-derived neurons and glia immediately and over a week following acute treatment. The administration of chronic LFS and DCS protocols individually following L-glutamate infusion significantly promoted the excitability of neurons as well as network synchrony, while the combination of LFS/DCS did not. qRT-PCR analysis revealed that LFS and DCS alone significantly up-regulated the expression of excitability and plasticity-related transcripts encoding N-methyl-D-aspartate (NMDA) receptor subunit (NR2A), brain-derived neurotrophic factor (BDNF) and Ras-related protein (RAB3A). In contrast, the simultaneous administration of LFS/DCS down-regulated BDNF and RAB3A expression. Our results demonstrate that LFS and DCS stimulation can modulate network maturation excitability and synchrony following the acute administration of an inhibitory dose of L-glutamate, and upregulate NR2A, BDNF and RAB3A gene expression. Our study also provides a novel framework for investigating the effects of electrical stimulation on neuronal responses and network formation and repair after traumatic brain injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-29069-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053382PMC
July 2018

Microfluidics in Malignant Glioma Research and Precision Medicine.

Adv Biosyst 2018 May 2;2(5). Epub 2018 Apr 2.

Regenerative Bioscience Center, ADS Complex, University of Georgia, 425 River Road, Athens, GA 30602-2771, USA.

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer that has no effective treatments and a prognosis of only 12-15 months. Microfluidic technologies deliver microscale control of fluids and cells, and have aided cancer therapy as point-of-care devices for the diagnosis of breast and prostate cancers. However, a few microfluidic devices are developed to study malignant glioma. The ability of these platforms to accurately replicate the complex microenvironmental and extracellular conditions prevailing in the brain and facilitate the measurement of biological phenomena with high resolution and in a high-throughput manner could prove useful for studying glioma progression. These attributes, coupled with their relatively simple fabrication process, make them attractive for use as point-of-care diagnostic devices for detection and treatment of GBM. Here, the current issues that plague GBM research and treatment, as well as the current state of the art in glioma detection and therapy, are reviewed. Finally, opportunities are identified for implementing microfluidic technologies into research and diagnostics to facilitate the rapid detection and better therapeutic targeting of GBM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adbi.201700221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5959050PMC
May 2018

Fast axial scanning for 2-photon microscopy using liquid lens technology.

Proc SPIE Int Soc Opt Eng 2017 Mar;10070

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

Scanning microscopy methods require movement of the focus in Z coordinates to produce an image of a 3-dimensional volume. In a typical imaging system, the optical setup is kept fixed and either the sample or the objective is translated with a mechanical stage driven by a stepper motor or a piezoelectric element. Mechanical Z scanning is precise, but its slow response and vulnerability to mechanical vibrations and stress make it disadvantageous to image dynamic, time-varying samples such as live cell structures. An alternative method less susceptible to these problems is to change the focal plane using conjugate optics. Deformable mirrors, acoustooptics, and electrically tunable lenses have been experimented with to achieve this goal and have attained very fast and precise Z-scanning without physically moving the sample. Here, we present the use of a liquid lens for fast axial scanning. Liquid lenses have a long functional life, high degree of phase shift, and low sensitivity to mechanical stress. They work on the principle of refraction at a liquid-liquid interface. At the boundary of a polar and an apolar liquid a spherical surface is formed whose curvature can be controlled by adjusting its relative wettability using electrowetting. We characterize the effects of the lens on attainable Z displacement, beam spectral characteristics, and pulse duration as compared with mechanical scanning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1117/12.2252992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918272PMC
March 2017

Sustained Release of Transforming Growth Factor-β1 from Platelet-Rich Chondroitin Sulfate Glycosaminoglycan Gels.

J Knee Surg 2018 May 23;31(5):410-415. Epub 2017 Jun 23.

Department of Small Animal Medicine and Surgery, Veterinary Teaching Hospital, University of Georgia, Athens, Georgia.

Activated platelet-rich plasma (PRP), also referred to as platelet-rich fibrin (PRF), has been used to augment numerous techniques of cartilage repair in the knee but does not always result in superior quality of repair tissue. One possible reason that PRF does not consistently result in excellent cartilage regeneration is the transiency of growth factor provision with PRF. The objective of this study was to compare the release of transforming growth factor (TGF)-β1 from PRF and from PRP combined with a novel chondroitin sulfate glycosaminoglycan (CS-GAG) gel. PRP was prepared from nine healthy dogs and split into two aliquots: one activated with bovine thrombin and calcium chloride (CaCl) to form PRF and the other aliquot was used to rehydrate a lyophilized CS-GAG gel. Both PRF and the CS-GAG gels were incubated in media for 13 days and media were collected, stored, and replaced every 48 hours and the concentration of TGF-β1 quantified in the media using an enzyme-linked immunosorbent assay. Concentrations of TGF-β1 in the media were up to three times greater with the CS-GAG gels and were significantly ( < 0.05) greater than with PRF on days 3, 5, 7, 9, and 13. Furthermore, TGF-β1 elution was still substantial at day 13 with the use of the CS-GAG gels. Additional in vitro work is warranted to characterize TGF-β1 elution from this CS-GAG gel with human PRP and to determine whether the use of these CS-GAG gels can augment cartilage repair in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/s-0037-1603801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916840PMC
May 2018

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsbiomaterials.6b00805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937277PMC
March 2017

Glioma Cell Invasion is Significantly Enhanced in Composite Hydrogel Matrices Composed of Chondroitin 4- and 4,6-Sulfated Glycosaminoglycans.

J Mater Chem B 2016 26;4(36):6052-6064. Epub 2016 Aug 26.

Regenerative Bioscience Center, ADS Complex, University of Georgia, Athens, Georgia.

Glioblastoma multiforme (GBM) is the most aggressive form of astrocytoma accounting for a majority of primary malignant brain tumors in the United States. Chondroitin sulfate proteoglycans (CSPGs) and their glycosaminoglycan (GAG) side chains are key constituents of the brain extracellular matrix (ECM) implicated in promoting tumor invasion. However, the mechanisms by which sulfated CS-GAGs promote brain tumor invasion are currently unknown. We hypothesize that glioma cell invasion is triggered by the altered sulfation of CS-GAGs in the tumor extracellular environment, and that this is potentially mediated by independent mechanisms involving CXCL12/CXCR4 and LAR signaling respectively. This was tested by encapsulating the human glioma cell line U87MG-EGFP into monosulfated (4-sulfated; CS-A), composite (4 and 4,6-sulfated; CS-A/E), unsulfated hyaluronic acid (HA), and unsulfated agarose (AG; polysaccharide) hydrogels within microfluidics-based choice assays. Our results demonstrated the enhanced preferential cell invasion into composite hydrogels, when compared to other hydrogel matrices (p<0.05). Haptotaxis assays demonstrated the significantly (p<0.05) faster migration of U87MG-EGFP cells in CXCL12 containing CS-GAG hydrogels when compared to other hydrogel matrices containing the same chemokine concentration. This is likely due to the significantly (p<0.05) greater affinity of composite CS-GAGs to CXCL12 over other hydrogel matrices. Results from qRT-PCR assays further demonstrated the significant (p<0.05) upregulation of the chemokine receptor CXCR4, and the CSPG receptor LAR in glioma cells within CS-GAG hydrogels compared to control hydrogels. Western blot analysis of cell lysates derived from glioma cells encapsulated in different hydrogel matrices further corroborate qRT-PCR results, and indicate the presence of a potential variant of LAR that is selectively expressed only in glioma cells encapsulated in CS-GAG hydrogels. These results suggest that sulfated CS-GAGs may directly induce enhanced invasion and haptotaxis of glioma cells associated with aggressive brain tumors via distinct mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/C6TB01083KDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312825PMC
August 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.bioconjchem.5b00397DOI Listing
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/nn505980zDOI Listing
February 2015

Intracortical recording interfaces: current challenges to chronic recording function.

ACS Chem Neurosci 2015 Jan 14;6(1):68-83. Epub 2015 Jan 14.

Regenerative Bioscience Center, ADS Complex, The University of Georgia , Athens, Georgia 30602-2771, United States.

Brain Computer Interfaces (BCIs) offer significant hope to tetraplegic and paraplegic individuals. This technology relies on extracting and translating motor intent to facilitate control of a computer cursor or to enable fine control of an external assistive device such as a prosthetic limb. Intracortical recording interfaces (IRIs) are critical components of BCIs and consist of arrays of penetrating electrodes that are implanted into the motor cortex of the brain. These multielectrode arrays (MEAs) are responsible for recording and conducting neural signals from local ensembles of neurons in the motor cortex with the high speed and spatiotemporal resolution that is required for exercising control of external assistive prostheses. Recent design and technological innovations in the field have led to significant improvements in BCI function. However, long-term (chronic) BCI function is severely compromised by short-term (acute) IRI recording failure. In this review, we will discuss the design and function of current IRIs. We will also review a host of recent advances that contribute significantly to our overall understanding of the cellular and molecular events that lead to acute recording failure of these invasive implants. We will also present recent improvements to IRI design and provide insights into the futuristic design of more chronically functional IRIs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cn5002864DOI Listing
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/micronano.2015.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258041PMC
June 2015

Chondroitin sulfate glycosaminoglycans for CNS homeostasis-implications for material design.

Curr Med Chem 2014 ;21(37):4257-81

Regenerative Bioscience Center, Edgar L. Rhodes Center for Animal and Dairy Sciences, The University of Georgia, Athens, GA 30602-2771, USA.

Chondroitin sulfate proteoglycans (CSPGs) are complex biomolecules that are known to facilitate patterning of axonal direction and cell migration during the early growth and development phase of the mammalian central nervous system (CNS). In adults, they continue to control neuronal plasticity as major constituents of the "peri-neuronal nets" (PNNs) that surround adult CNS neurons. CSPGs are also barrier-forming molecules that are selectively upregulated by invading reactive astroglia after injury to the CNS, and are responsible for the active repulsion of regenerating neurons post-injury. Recent evidence however suggests that the diverse sulfated glycosaminoglycan (GAG) side chains attached to CSPGs are key components that play paradoxical roles in influencing nerve regeneration post-injury to the CNS. Sulfated GAG repeats attached to the CSPG core protein help mediate cell migration, neuritogenesis, axonal pathfinding, and axonal repulsion by directly trapping and presenting a whole host of growth factors to cells locally, or by binding to specific membrane bound proteins on the cell surface to influence cellular function. In this review, we will present the current gamut of interventional strategies used to bridge CNS deficits, and discuss the potential advantages of using sulfated GAG based biomaterials to facilitate the repair and regeneration of the injured CNS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/0929867321666140815124447DOI Listing
June 2015

Molecular sequelae of topographically guided peripheral nerve repair.

Ann Biomed Eng 2014 Jul 20;42(7):1436-55. Epub 2013 Dec 20.

Neurological Biomaterials and Cancer Therapeutics Laboratory, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University School of Medicine, UA Whitaker Building, 313 Ferst Drive, Atlanta, GA, 30332-0535, USA.

Peripheral nerve injuries cause severe disability with decreased nerve function often followed by neuropathic pain that impacts the quality of life. Even though use of autografts is the current gold standard, nerve conduits fabricated from electrospun nanofibers have shown promise to successfully bridge critical length nerve gaps. However, in depth analysis of the role of topographical cues in the context of spatio-temporal progression of the regenerative sequence has not been elucidated. Here, we explored the influence of topographical cues (aligned, random, and smooth films) on the regenerative sequence and potential to successfully support nerve regeneration in critical size gaps. A number of key findings emerged at the cellular, cytokine and molecular levels from the study. Higher quantities of IL-1α and TNF-α were detected in aligned fiber based scaffolds. Differential gene expression of BDNF, NGFR, ErbB2, and ErbB3 were observed suggesting a role for these genes in influencing Schwann cell migration, myelination, etc. that impact the regeneration in various topographies. Fibrin matrix stabilization and arrest of nerve-innervated muscle atrophy was also evident. Taken together, our data shed light on the cascade of events that favor regeneration in aligned topography and should stimulate research to further refine the strategy of nerve regeneration using topographical cues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10439-013-0960-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516464PMC
July 2014

Relationship between intracortical electrode design and chronic recording function.

Biomaterials 2013 Nov 26;34(33):8061-74. Epub 2013 Jul 26.

Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology and Emory University School of Medicine, 313 Ferst Drive, Atlanta, GA 30332-0535, USA.

Intracortical electrodes record neural signals directly from local populations of neurons in the brain, and conduct them to external electronics that control prosthetics. However, the relationship between electrode design, defined by shape, size and tethering; and long-term (chronic) stability of the neuron-electrode interface is poorly understood. Here, we studied the effects of various commercially available intracortical electrode designs that vary in shape (cylindrical, planar), size (15 μm, 50 μm and 75 μm), and tethering [electrode connections to connector with (tethered) and without tethering cable (untethered)] using histological, transcriptomic, and electrophysiological analyses over acute (3 day) and chronic (12 week) timepoints. Quantitative analysis of histological sections indicated that Michigan 50 μm (M50) and Michigan tethered (MT) electrodes induced significantly (p < 0.01) higher glial scarring, and lesser survival of neurons in regions of blood-brain barrier (BBB) breach when compared to microwire (MW) and Michigan 15 μm (M15) electrodes acutely and chronically. Gene expression analysis of the neurotoxic cytokines interleukin (Il)1 (Il1α, Il1β), Il6, Il17 (Il17a, Il17b, Il17f), and tumor necrosis factor alpha (Tnf) indicated that MW electrodes induced significantly (p < 0.05) reduced expression of these transcripts when compared to M15, M50 and FMAA electrodes chronically. Finally, electrophysiological assessment of electrode function indicated that MW electrodes performed significantly (p < 0.05) better than all other electrodes over a period of 12 weeks. These studies reveal that intracortical electrodes with smaller size, cylindrical shape, and without tethering cables produce significantly diminished inflammatory responses when compared to large, planar and tethered electrodes. These studies provide a platform for the rational design and assessment of chronically functional intracortical electrode implants in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2013.07.016DOI Listing
November 2013

The impact of chronic blood-brain barrier breach on intracortical electrode function.

Biomaterials 2013 Jul 2;34(20):4703-13. Epub 2013 Apr 2.

Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology, Emory University School of Medicine, Atlanta, GA 30332-0535, USA.

Brain-computer interfaces (BCIs) have allowed control of prosthetic limbs in paralyzed patients. Unfortunately, the electrodes of the BCI that interface with the brain only function for a short period of time before the signal quality on these electrodes becomes substantially diminished. To truly realize the potential of BCIs, it is imperative to have electrodes that function chronically. In order to elucidate the physiological determinants of a chronically functional neural interface, we studied the role of the blood-brain barrier (BBB) in electrode function, because it is a key mediator of neuronal hemostasis. We monitored the status of the BBB and the consequences of BBB breach on electrode function using non-invasive imaging, electrophysiology, genomic, and histological analyses. Rats implanted with commercially available intracortical electrodes demonstrated an inverse correlation between electrode performance and BBB breach over a period of 16 weeks. Genomic analysis showed that chronically functional electrodes elicit an enhanced wound healing response. Conversely, in poorly functioning electrodes, chronic BBB breach led to local accumulation of neurotoxic factors and an influx of pro-inflammatory myeloid cells, which negatively affect neuronal health. These findings were further verified in a subset of electrodes with graded electrophysiological performance. In this study, we determine the mechanistic link between intracortical electrode function and failure. Our results indicate that BBB status is a critical physiological determinant of intracortical electrode function and can inform future electrode design and biochemical intervention strategies to enhance the functional longevity of BCIs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2013.03.007DOI Listing
July 2013

The upregulation of specific interleukin (IL) receptor antagonists and paradoxical enhancement of neuronal apoptosis due to electrode induced strain and brain micromotion.

Biomaterials 2012 Sep 6;33(26):5983-96. Epub 2012 Jun 6.

Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology and Emory University School of Medicine, Atlanta, 313 Ferst Drive, GA 30332-0535, USA.

The high mechanical mismatch between stiffness of silicon and metal microelectrodes and soft cortical tissue, induces strain at the neural interface which likely contributes to failure of the neural interface. However, little is known about the molecular outcomes of electrode induced low-magnitude strain (1-5%) on primary astrocytes, microglia and neurons. In this study we simulated brain micromotion at the electrode-brain interface by subjecting astrocytes, microglia and primary cortical neurons to low-magnitude cyclical strain using a biaxial stretch device, and investigated the molecular outcomes of induced strain in vitro. In addition, we explored the functional consequence of astrocytic and microglial strain on neural health, when they are themselves subjected to strain. Quantitative real-time PCR array (qRT-PCR Array) analysis of stretched astrocytes and microglia showed strain specific upregulation of an Interleukin receptor antagonist - IL-36Ra (previously IL-1F5), to ≈ 1018 and ≈ 236 fold respectively. Further, IL-36Ra gene expression remained unchanged in astrocytes and microglia treated with bacterial lipopolysaccharide (LPS) indicating that the observed upregulation in stretched astrocytes and microglia is potentially strain specific. Zymogram and western blot analysis revealed that mechanically strained astrocytes and microglia upregulated matrix metalloproteinases (MMPs) 2 and 9, and other markers of reactive gliosis such as glial fibrillary acidic protein (GFAP) and neurocan when compared to controls. Primary cortical neurons when stretched with and without IL-36Ra, showed a ≈ 400 fold downregulation of tumor necrosis factor receptor superfamily, member 11b (TNFRSF11b). Significant upregulation of members of the caspase cysteine proteinase family and other pro-apoptotic genes was also observed in the presence of IL-36Ra than in the absence of IL-36Ra. Adult rats when implanted with microwire electrodes showed upregulation of IL-36Ra (≈ 20 fold) and IL-1Ra (≈ 1500 fold) 3 days post-implantation (3 DPI), corroborating in vitro results, although these transcripts were drastically down regulated by ≈ 20 fold and ≈ 1488 fold relative to expression levels 3 DPI, at the end of 12 weeks post-implantation (12 WPI). These results demonstrate that IL receptor antagonists may be negatively contributing to neuronal health at acute time-points post-electrode implantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.biomaterials.2012.05.021DOI Listing
September 2012

Anti-invasive adjuvant therapy with imipramine blue enhances chemotherapeutic efficacy against glioma.

Sci Transl Med 2012 Mar;4(127):127ra36

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

The invasive nature of glioblastoma (GBM) represents a major clinical challenge contributing to poor outcomes. Invasion of GBM into healthy tissue restricts chemotherapeutic access and complicates surgical resection. Here, we test the hypothesis that an effective anti-invasive agent can "contain" GBM and increase the efficacy of chemotherapy. We report a new anti-invasive small molecule, Imipramine Blue (IB), which inhibits invasion of glioma in vitro when tested against several models. IB inhibits NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-mediated reactive oxygen species generation and alters expression of actin regulatory elements. In vivo, liposomal IB (nano-IB) halts invasion of glioma, leading to a more compact tumor in an aggressively invasive RT2 syngeneic astrocytoma rodent model. When nano-IB therapy was followed by liposomal doxorubicin (nano-DXR) chemotherapy, the combination therapy prolonged survival compared to nano-IB or nano-DXR alone. Our data demonstrate that nano-IB-mediated containment of diffuse glioma enhanced the efficacy of nano-DXR chemotherapy, demonstrating the promise of an anti-invasive compound as an adjuvant treatment for glioma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.3003016DOI Listing
March 2012

Photocrosslinkable chitosan based hydrogels for neural tissue engineering.

Soft Matter 2012 Feb 23;8(6):1964-1976. Epub 2011 Dec 23.

Neurological Biomaterials and Cancer Therapeutics Laboratory, USA.

Hydrogel based scaffolds for neural tissue engineering can provide appropriate physico-chemical and mechanical properties to support neurite extension and facilitate transplantation of cells by acting as 'cell delivery vehicles'. Specifically, gelling systems such as photocrosslinkable hydrogels can potentially conformally fill irregular neural tissue defects and serve as stem cell delivery systems. Here, we report the development of a novel chitosan based photocrosslinkable hydrogel system with tunable mechanical properties and degradation rates. A two-step synthesis of amino-ethyl methacrylate derivitized, degradable, photocrosslinkable chitosan hydrogels is described. When human mesenchymal stem cells were cultured in photocrosslinkable chitosan hydrogels, negligible cytotoxicity was observed. Photocrosslinkable chitosan hydrogels facilitated enhanced neurite differentiation from primary cortical neurons and enhanced neurite extension from dorsal root ganglia (DRG) as compared to agarose based hydrogels with similar storage moduli. Neural stem cells (NSCs) cultured within photocrosslinkable chitosan hydrogels facilitated differentiation into tubulin positive neurons and astrocytes. These data demonstrate the potential of photocrosslinked chitosan hydrogels, and contribute to an increasing repertoire of hydrogels designed for neural tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c1sm06629cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969809PMC
February 2012

Targeted downregulation of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase significantly mitigates chondroitin sulfate proteoglycan-mediated inhibition.

Glia 2011 Jun 31;59(6):981-96. Epub 2011 Mar 31.

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

Chondroitin sulfate-4,6 (CS-E) glycosaminoglycan (GAG) upregulation in astroglial scars is a major contributor to chondroitin sulfate proteoglycan (CSPG)-mediated inhibition [Gilbert et al. (2005) Mol Cell Neurosci 29:545–558]. However, the role of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S6ST) catalyzed sulfation of CS-E, and its contribution to CSPG-mediated inhibition of CNS regeneration remains to be fully elucidated. Here, we used in situ hybridization to show localized upregulation of GalNAc4S6ST mRNA after CNS injury. Using in vitro spot assays with immobilized CS-E, we demonstrate dose-dependent inhibition of rat embryonic day 18 (E18) cortical neurons. To determine whether selective downregulation of CS-E affected the overall inhibitory character of extracellular matrix produced by reactive astrocytes, single [against (chondroitin 4) sulfotransferase 11 (C4ST1) or GalNAc4S6ST mRNA] or double [against C4ST1 and GalNAc4S6ST mRNA] siRNA treatments were conducted and assayed using quantitative real-time polymerase chain reaction and high-performance liquid chromatography to confirm the specific downregulation of CS-4S GAG (CS-A) and CS-E. Spot and Bonhoeffer stripe assays using astrocyte-conditioned media from siRNA-treated rat astrocytes showed a significant decrease in inhibition of neuronal attachment and neurite extensions when compared with untreated and TGF-treated astrocytes. These findings reveal that selective attenuation of CS-E via siRNA targeting of GalNAc4S6ST significantly mitigates CSPG-mediated inhibition of neurons, potentially offering a novel intervention strategy for CNS injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.21170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3077466PMC
June 2011

Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis.

PLoS One 2011 Mar 1;6(3):e17606. Epub 2011 Mar 1.

Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, Tennessee, United States of America.

Development of insect resistance is one of the main concerns with the use of transgenic crops expressing Cry toxins from the bacterium Bacillus thuringiensis. Identification of biomarkers would assist in the development of sensitive DNA-based methods to monitor evolution of resistance to Bt toxins in natural populations. We report on the proteomic and genomic detection of reduced levels of midgut membrane-bound alkaline phosphatase (mALP) as a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to susceptible larvae. Reduced levels of H. virescens mALP protein (HvmALP) were detected by two dimensional differential in-gel electrophoresis (2D-DIGE) analysis in Cry-resistant compared to susceptible larvae, further supported by alkaline phosphatase activity assays and Western blotting. Through quantitative real-time polymerase chain reaction (qRT-PCR) we demonstrate that the reduction in HvmALP protein levels in resistant larvae are the result of reduced transcript amounts. Similar reductions in ALP activity and mALP transcript levels were also detected for a Cry1Ac-resistant strain of H. armigera and field-derived strains of S. frugiperda resistant to Cry1Fa. Considering the unique resistance and cross-resistance phenotypes of the insect strains used in this work, our data suggest that reduced mALP expression should be targeted for development of effective biomarkers for resistance to Cry toxins in lepidopteran pests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017606PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046977PMC
March 2011
-->