Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury

Abstract

Event Abstract Back to Event Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury Josna Joseph1*, Kazuhide Hayakawa2*, Motoichi Kurisawa3*, Li Shan Wang3*, Wanting Niu1 and Myron Spector1 1 VA Boston Healthcare System, Brigham&Women's Hospital, Harvard Medical School, Tissue Engineering, United States 2 Massachusetts General Hospital, Neuroprotection Research laboratory, Department of Neurology& Radiology, United States 3 Institute for Bioengineering and Nanotechnology, Singapore Introduction: The glial scar that is formed around residual cavities after certain spinal cord injuries is a physical and physiological impediment to neuronal regeneration. Gelatin–hydroxyphenyl propionic acid (Gtn-HPA), a novel injectable matrix with tuneable cross-linking properties[1], could provide the missing stroma in such defects, enabling migration of astrocytes from the glial scar into the matrix-filled defect and their guidance of neurons into the lesion[2]. The principal objective of this study was to evaluate astrocyte migration and activation in an in vitro ‘tissue simulant’ model using Gtn-HPA incorporating select growth factors. Materials and Methods: Cortical astrocytes were isolated from postnatal rats, and grown in DMEM high glucose medium. Astrocyte proliferation in Gtn-HPA gels at days 2, 4 and 7 was analysed by counting live cells stained by Calcein-AM under fluorescence microscopy. A three-dimensional migration assay model was set up in 3% agarose-coated 24-well culture plates by surrounding Gtn-HPA cores (1cm diameter) containing EGF (50 or 200 ng/ul) or TGF-β1 (50 or 200 ng/ul) with an annular tissue simulant of 2 wt. % Gtn-HPA gel (cross-linked with 0.1 U/ml horseradish peroxidase and 1.2 mM H202) containing 3*105 cells/ml. Cellular migration into the growth factor-laden inner core was analysed quantitatively by counting cells and measuring the migration distance (n=5 fields) from the gel interface to the farthest cell body (n=4 gels) under phase contrast microscopy. Immunostaining of 30-mm thick cryo sections of gel construct was carried out to detect the presence of GFAP-positive astrocytes. GFAP expression by migrated cells was quantified by Western blot by lysing the gel with collagenase 1 (500U/ml). The results were tested for statistical significance by ANOVA and post-hoc tests. Results and Discussion: Astrocytes migrated in response to chemotactic gradients in Gtn-HPA gels in contrast to the blank gels which displayed virtually no migration. The migration distance was significantly higher (p<0.001) in TGF-50 ng (TGFL) group when compared to EGF and blank Gtn-HPA control group (Fig.1). The number of cells infiltrating the inner core and GFAP expression was significantly higher in the EGF-50 ng (EGFL) group (Fig.2). This indicates that astrocytes migrate well in response to TGF-β1 and are activated by EGF at low concentration, whereas higher conc. of EGF has no specific effect on GFAP expression. Conclusion: This study shows that growth factor-laden Gtn-HPA hydrogels provide a supportive environment for astrocyte migration, proliferation and activation. Gtn-HPA hydrogels laden with optimum combination of EGF and TFG-β1 could effectively guide astrocyte infiltration and subsequent neurite outgrowth in a spinal cord injury defect with diminished glial scarring. J. Joseph acknowledges Fulbright Fellowship from United States India Educational Foundation. Gtn-HPA was provided by the Institute of Bioengineering and Nanotechnology, Singapore.