Traumatic brain injury (TBI) often results in debilitating loss of cognitive and motor function. TBI is characterized by a primary mechanical trauma followed by a persistent, inflammatory secondary injury cascade. Together, these lead to damage and death of neurons followed by the formation of an inhibitory lesion cavity, as well as a glial scar, which impedes regeneration. Although there are currently no fully restorative treatments for TBI, the delivery of brain derived neurotrophic factor (BDNF) has been shown to be beneficial in animal models of TBI; however its therapeutic potential is limited by rapid clearance in vivoand poor permeability across the blood-brain barrier. Neural stem cells (NSCs) hold promise as a replacement cell source; however NSCs directly transplanted to the site of brain injury suffer from poor viability and localization. Recently, short peptides that mimic aspects of the beneficial activity of native BDNF have been identified. We are developing a therapeutic hydrogel system by covalently grafting one of these peptides, IKRG, to type-I collagen. Herein, we describe development of this system, and demonstrate its bioactivity with neurons and NSCs.
IKRG Peptide Grafting Type-I bovine collagen (Elastin Products Company, Owensville, MO) was reconstituted in 0.02N acetic acid at 3mg/mL. IKRG peptides were synthesized commercially (Genscript, Piscataway, NJ), dissolved in MES buffer along with 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), and allowed to react for 15 minutes at 37°C to activate the carboxyl groups on the peptide. The activated peptide was added to the 3 mg/mL collagen suspension and allowed to incubate at 4°C overnight on a rotator. The resulting solution was dialyzed to remove un-grafted peptide, lyophilized, and reconstituted at 3mg/mL in 0.02N acetic acid. Hydrogel Preparation Buffered hydrogel solutions from native or grafted collagen were prepared using the following ratios: 20µL HEPES:130µL 0.15N NaOH:100µL 10x MEM: 56µL M199: 10µL L-glutamine: 10µL penicillin/streptomycin: 677µL 3.0mg/mL collagen solution. Hydrogels with varied peptide concentration were created by admixing grafted and native collagen hydrogel suspensions at target ratios. Hydrogel suspensions were placed in multiwall plates and allowed to self-assemble at 37°C for one hour. Cerebellar Neuron Culture Cerebellar Granule Neurons (CGNs) were isolated from day 6 rat pups and cultured on collagen hydrogel conditions at ~50,000 cells/cm2 in Neurobasal Medium (Life Technologies) containing 0.5% L-glutamine, 1% penicillin/streptomycin, 2% B27 supplement, and 1% potassium chloride. IKRG and BDNF were included at 1µM and 20ng/mL respectively in the requisite controls. Neural Stem Cell Culture Induced pluripotent stem cell derived neural stem cells (NSCs) were cultured in a 50:50 mixture of neruobasal medium:DMEM F-12+Glutamax containing 0.5% N2 supplement, 1% B27 supplement, 0.5% penicillin/streptomycin and supplemented with 2ng/mL basic fibroblast growth factor. NSCs were seeded onto hydrogels at ~12,500 cells/cm2. Cellular Viability Phosphate buffered saline containing 0.5µL/mL and 1µL/mL of Calcien AM and Ethidium Homodimer (Invitrogen) respectively, were added to cultures and incubated for 45 minutes at 37°C. Cultures were observed with fluorescent microscopy and counted in ImageJ. Immunocytochemistry: CGN cultures were stained immunohistochemically for β-III tubulin (TUJ1) and DAPI while NSC cultures were stained for DAPI, Nestin, and TUJ1, before being observed with fluorescent microscopy. Neurite outgrowth of CGNs was quantified from TUJ1 images using ImageJ.
Results and Discussion
CGNs cultured on IKRG grafted collagen hydrogels demonstrated longer neurite outgrowth than native collagen controls in a dose-dependent manner. CGNs cultured on IKRG-grafted hydrogels demonstrated average neurite length of 216.2 µm (+/- 34.4 µm) compared to 100.7µm, (+/- 9.9 µm) on native collagen. Growth on collagen with lower concentrations of grafted peptide fell between these two conditions. The increase in neurite length compared well with controls where soluble BDNF (average neurite length 204.5 µm +/- 21.2 µm) or soluble IKRG (average neurite length 249.0 µm +-/- 50.9 µm) was added. These results confirm that the IKRG peptides remain bioactive following covalent coupling to collagen with potency comparable to native, soluble BDNF. Viability of both CGNs and NSCs on IKRG grafted collagen hydrogels was similar to conditions with soluble IKRG peptide and BDNF as well as native collagen. Preliminary work shows that NSCs cultured on IKRG grafted hydrogels were positive for βIII tubulin by DIV7, demonstrating that IKRG grafted hydrogels may support the neuronal differentiation of NSCs even in the absence of soluble factors.
Through this work, we have confirmed that IKRG peptides retain their bioactivity when covalently coupled to collagen with in vitro potency comparable to the soluble addition of BDNF. Ongoing work will assess the ability of this hydrogel system to enhance neuronal differentiation of NSCs and preserve neuronal viability using in vitromodels of secondary brain injury such as glutamate induced excitotoxicity.
This work is supported by a Graduate Fellowship from the New Jersey Commission on Brain Injury Research (CBIR14FEL004)