- Department of Neurosurgery, Baylor College of Medicine, Houston, Texas, USA
- Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA
- Department of Neurology, Baylor College of Medicine, Houston, Texas, USA
- Interdepartmental Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA
- Division of Pediatric Neurosurgery, Texas Children's Hospital, Houston, Texas, USA
- Department of Chemistry, Rice University, Houston, Texas, USA
- Department of Chemistry and Materials Science and NanoEngineering, Rice University, Houston, Texas, USA
- Research and Tissue Support Services Core Laboratory, Texas Children's Cancer and Hematology Services, Houston, Texas, USA
- Center for Translational Research in Inflammatory Diseases, Michael E. DeBakey VA Medical Center, Houston, Texas, USA
Department of Neurosurgery, Baylor College of Medicine, Houston, Texas, USA
Division of Pediatric Neurosurgery, Texas Children's Hospital, Houston, Texas, USA
DOI:10.4103/2152-7806.188905Copyright: © 2016 Surgical Neurology International This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
How to cite this article: Palejwala AH, Fridley JS, Mata JA, G. Samuel EL, Luerssen TG, Perlaky L, Kent TA, Tour JM, Jea A. Biocompatibility of reduced graphene oxide nanoscaffolds following acute spinal cord injury in rats. Surg Neurol Int 23-Aug-2016;7:75
How to cite this URL: Palejwala AH, Fridley JS, Mata JA, G. Samuel EL, Luerssen TG, Perlaky L, Kent TA, Tour JM, Jea A. Biocompatibility of reduced graphene oxide nanoscaffolds following acute spinal cord injury in rats. Surg Neurol Int 23-Aug-2016;7:75. Available from: http://surgicalneurologyint.com/surgicalint_articles/biocompatibility-reduced-graphene-oxide-nanoscaffolds-following-acute-spinal-cord-injury-rats/
Background:Graphene has unique electrical, physical, and chemical properties that may have great potential as a bioscaffold for neuronal regeneration after spinal cord injury. These nanoscaffolds have previously been shown to be biocompatible in vitro; in the present study, we wished to evaluate its biocompatibility in an in vivo spinal cord injury model.
Methods:Graphene nanoscaffolds were prepared by the mild chemical reduction of graphene oxide. Twenty Wistar rats (19 male and 1 female) underwent hemispinal cord transection at approximately the T2 level. To bridge the lesion, graphene nanoscaffolds with a hydrogel were implanted immediately after spinal cord transection. Control animals were treated with hydrogel matrix alone. Histologic evaluation was performed 3 months after the spinal cord transection to assess in vivo biocompatibility of graphene and to measure the ingrowth of tissue elements adjacent to the graphene nanoscaffold.
Results:The graphene nanoscaffolds adhered well to the spinal cord tissue. There was no area of pseudocyst around the scaffolds suggestive of cytotoxicity. Instead, histological evaluation showed an ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells around the graphene nanoscaffolds.
Conclusions:Graphene is a nanomaterial that is biocompatible with neurons and may have significant biomedical application. It may provide a scaffold for the ingrowth of regenerating axons after spinal cord injury.
Keywords: Biocompatibility, cytotoxicity, graphene, nanomedicine, neuron, spinal cord injury
Few options are available for the treatment of spinal cord injury (SCI), despite years of research on the subject. Supportive medical care and early surgical decompression, when applicable,[
A consequence of SCI is the formation of scar tissue and posttraumatic microcystic myelomalacia.[
Development of a bioscaffold that assists with neural tissue regeneration and the prevention/bypass of scar formation is an ongoing area of research. Graphene, a two-dimensional (2D) sheet of sp2-hybridized graphitic carbon,[
Two-dimensional graphene films have been used for studying neuronal growth in vitro,[
Given graphene's potential as a nanoscaffold that might physically support, electrically stimulate, positionally inform, and organize the 3D cytoarchitecture required for axonal regeneration, there is a need to assess the interaction and bioreactivity of this nanomaterial with mammalian neuronal cells in a rat model for SCI. At this early phase of our investigation of graphene as an adequate scaffold in the setting of SCI, we are not ready to compare our nanoscaffold with that of other authors.[
MATERIALS AND METHODS
Graphene oxide scaffold preparation
GO (38 mg, produced by the method of Marcano et al.[
The morphology of the resulting hydrogel was characterized by scanning electron microscopy (SEM) after lyophilization. The porous structure was formed by crosslinking graphene sheets. The pore sizes ranged from hundreds of nanometers to several micrometers [
Scaffold implantation and animal care
A hemispinal cord transection was selected as a model for evaluating in vivo the effect of graphene implantation on SCI immediately after creating the lesion. Twenty (19 male, 1 female) 8-week-old Wistar rats (Velaz, Ltd., Prague, Czech Republic), each weighing 300–400 g, were used. Eight hours prior to the surgery, food was withheld, and carprofen (analgesic tablets) was administered. Anesthesia was induced by means of inhaled 2% isofluorane. The skin over the upper thoracic spine was shaved and prepped. A laminectomy was performed at approximately T2 under a surgical microscope using aseptic technique. The dura was opened; a 2 mm-wide hemi-segment of spinal cord was excised from the left side, producing the SCI. The segment was examined using a surgical microscope to ensure that no remaining tissue was left.
In 10 animals, a hydrogel matrix (HydroGel™, Portland, Maine) was laid onto the injured spinal cord and open dura. This was followed by approximation of paraspinal muscle with sutures (Vicryl, Johnson and Johnson, Somerville, New Jersey) and skin closure with skin staples (MultiFire Premium, Covidien, Dublin, Ireland). These animals served as the control group.
In 10 animals, we inserted an approximately 2 × 2 × 2-mm block of the reduced GO scaffold with an overlying layer of hydrogel matrix. This was followed by muscle and skin closure. These animals served as the treatment group.
In both groups, bladder expression was performed until recovery of sphincter control; enrofloxacin (5–10 mg/kg) was administered subcutaneously for 7 days to prevent urinary infection. Animals were kept in cages with food and water ad lib. Pain control was provided for 5 days after surgery with buprenorphine (0.05–0.5 mg/kg), banamine (2–4 mg/kg), and/or rimadyl tablets. This study was performed in accordance with the guidelines of our Institutional Animal Care and Use Committee (IACUC) and was approved by the Baylor College of Medicine Institutional Review Board (Protocol #AN-6016).
No unexpected deaths occurred in follow-up before intentional sacrifice of the animals in the present experimental protocol. In prior study iterations with a complete transection of the spinal cord, we had experienced an unacceptably high mortality rate secondary to permanent bladder dysfunction and presumed urinary tract infection/urosepsis.
Tissue processing and histology
The animals were sacrificed 3 months after the surgery using CO2 inhalation. A 3 cm-long segment of the thoracic spinal column with spinal cord (including injury epicenter with implanted graphene scaffold and/or hydrogel) was carefully dissected out and left overnight in 10% buffered neutral pH formalin. The bone was removed; the spinal cord itself was postfixed in the same fixative for 24 h. Each spinal cord was embedded in paraffin and cut in the coronal plane into 10 μm-thick sections on a cryostat. The sections were stained with Hematoxylin and eosin (H and E), Luxol fast blue, and cresyl violet using standard protocols. For immunohistochemical studies, the following primary antibodies and dilutions were used: GFAP-Cy3 (1:200, Sigma-Aldrich, St. Louis, Missouri) to identify astrocytes, NF 160 (1:200, Sigma-Aldrich, St. Louis, Missouri) to identify neurofilaments, p75 (1:100, Chemicon International, Temecula, California) to identify Schwann cells, RECA-1 (1:50, Abcam, Cambridge) to identify endothelial cells of blood vessels, ED-1 (1:100, Invitrogen, Waltham, Massachusetts) to identify macrophages, CS-56 (1:50, Sigma-Aldrich, St. Louis, Missouri) to identify chondroitin sulfate, and CD4 (1:800, Abcam, Cambridge, UK). Alexa Fluor 488 goat anti-rabbit IgG (1:200, Invitrogen, Waltham, Massachusetts), IgM Cy3 (1:100, Chemicon International, Temecula, California), and Alexa Fluor 594 goat anti-rabbit IgG (1:500, Invitrogen, Waltham, Massachusetts) were used as secondary antibodies. To confirm the presence of cells, immunostained sections were additionally stained with DAPI (0.4 ug/ml, Chemicon International, Temecula, California) to identify all the cell nuclei.
The authors acknowledge financial support from the U.S. Army Telemedicine Advanced Technology Research Center (TATRC)/Alliance for NanoHealth (Grant No. W81XWH-09-2-0139; A.J. and J.M.T.); AOSpine North America Young Investigator Research Award (A.J.) and Texas Children's Hospital Department of Surgery Seed Research Fund (A.J.). For the remaining authors, none was received.
Comparison of control and treatment groups
We compared the results of reduced GO hydrogel scaffold implantation in the treated group with the hydrogel-only implantation control group. The treatment group showed good graphene scaffold integration inside the lesion site [
Representative photomicrographs showing spinal cord injury development after hemispinal cord transection at the T2 level (a) with reduced graphene oxide nanoscaffold performed immediately after transection and (b) without nanoscaffold implantation (control group). Notice the area devoid of tissue (arrow) at the lesion site in the control slide, suggesting possible pseudocyst formation. By contrast, cell proliferation (asterisk) is exuberant with implantation of the nanoscaffold, and no cavity is evident. Hematoxylin and eosin bar = 2 mm
Histological evaluation of reduced graphene oxide scaffold integration
Three months after SCI, connective tissue elements, such as fibroblasts, collagen, blood vessels, and chondroitin sulfate, were densely adherent in and around the graphene nanoscaffold [
Photomicrographs. CS-56 demonstrates chondroitin sulfate around the nanoscaffold: (a) Phase contrast, (b) immunostained, and (c) DAPI superimposed. RECA-1 demonstrates the presence of blood vessels: (d) Phase contrast, (e) immunostained, and (f) DAPI superimposed. NF-160-g488 shows neurofilaments: (g) Phase contrast, (h) immunostained, and (i) DAPI superimposed. P75 shows Schwann cells: (j) Phase contrast, (k) immunostained, and (l) DAPI superimposed. GFAP-Cy3 demonstrates astrocytes: (m), Phase contrast, (n) immunostained, and (o) DAPI superimposed. Bar = 150 μm
Neurofilaments grew toward the nanoscaffold at the tissue-implant border [
Histologic observation of the lesion site in the hydrogel-only matrix showed a large area devoid of tissue [
Tissue engineering and nanotechnology represent a promising approach for modulating the perilesional inhibitory environment in SCI to facilitate recovery and axonal growth. Other authors have used a variety of biomaterials and nanoparticles in SCI as an injectable nonstructured delivery vehicle after injury, with or without seeding with neural stem cells. Promising materials for scaffolds have included natural polymers, such as collagen, agarose, chitosan, and synthetic polymers such as poly(lactide-co-glycolide)/polyethylene glycol.[
Collagen, a widely used biomaterial, is both biocompatible and biodegradable.[
Agarose, a biocompatible material that can withstand biodegradation over a month in vivo,[
Chitosan is a naturally available polysaccharide found in the exoskeletons of crustaceans and insects. After being filled with type I collagen, a chitosan tube was implanted in a transected spinal cord.[
Poly(lactic-co-glycolic acid) (PLGA), a synthetic copolymer of polylactic acid and polyglycolic acid, is biocompatible and biodegradable.[
The graphene nanoscaffold, when implanted into the site of injury, provided a surface for growth, attachment, and survival of tissue [
Representative phase contrast photomicrographs illustrate structural regeneration of spinal cord tissue using a nanoscaffold. The incompletely transected spinal cord is bridged using a reduced graphene oxide scaffold for tissue ingrowth and cell infiltration. (a), Spinal cord adheres well to the nanoscaffold. (b), Loose connective tissue forms between the spinal cord tissue and the reduced graphene oxide scaffold. Bar = 150 μm
We were able to confirm neuronal regeneration with NF-160-stained slides, which demonstrated neurofilament growth that paralleled the contour of the graphene nanoscaffold [
The presence of connective elements necessary for neuronal regeneration was detected around the graphene nanoscaffold. Chondroitin sulfate, stained with CS-56, was observed, with growth centered at the tissue-implant border [
Astrocytes, stained with GFAP-Cy3, were observed to have dense growth at the injury site, specifically bordering newly regenerated tissue around the graphene nanoscaffold [
The use of artificial implants in SCI raises concerns for possible inflammatory reactions toward the material. In our study, we observed cellular infiltration close to the graphene nanoscaffold of predominantly macrophages, as seen on H and E-stained sections and confirmed with ED-1 immunohistochemical stains, along with a few lymphocytes.
Combining scaffold implantation with the use of neurotrophic factors or stem cell treatment may lead to improved results. Loh et al.[
Our work to date has determined that graphene-based nanomaterials represent an additional promising group of bioscaffolds with potential to further advance SCI research. These nanoscaffolds have previously been shown to be biocompatible in vitro;[
Our study was qualitative in nature rather than quantitative. For example, because of artifact from tissue fixation and processing, we were unable to quantitatively evaluate the size of the perilesional pseudocyst in any experimental cohort. A qualitative study has many strengths: Playing an important role in suggesting possible relationships, causes, effects, and dynamic processes; examining forms of knowledge that otherwise might be unavailable through statistical analysis, thereby gaining new insight; and adding “flesh and blood” to the analysis of clinical implications. Nonetheless, qualitative research has significant drawbacks. The problem of adequate validity or reliability is a major criticism. Because of the subjective nature of qualitative data and its origin in single contexts, it is difficult to apply conventional standards of reliability and validity. Contexts, situations, events, conditions, and interactions cannot be replicated to any extent, nor can generalizations be made to a wider context other than the one studied with any confidence. In future study iterations, our methodology will include ways to quantitatively assess our data, including that contained in Figures
Furthermore, our methodology did not include Luxol fast blue staining to evaluate the preserved gray and white matter and the perilesional cavity regions or GAP43 to show axonal elongation. A number of immunostaining methods could have been used; however, there is precedent[
Another weakness of our analysis is that we did not assess functional outcomes in our control and experimental animals. This deficiency will represent an arm of our investigation in future studies in both hemispinal and complete spinal cord transection models.
Our study showed the biocompatibility of an immobilized graphene-structured surface for direct neuronal interface in vivo. The 3D structure of reduced GO hydrogel allowed the growth of blood vessels, neurofilaments, and Schwann cells on its surface. Given its low toxicity when compared with other nanomaterials, graphene has significant potential as a key material in neuronal interface studies. Graphene's ability to carry neuroregenerative biomolecules, electrical conductivity, and neurocompatibility suggest the need for further investigation as a nanoscaffold in the treatment of SCI.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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