An injectable, calcium responsive composite hydrogel for the treatment of acute spinal cord injury

Abstract

Immediately following spinal cord injury, further injury can occur through several secondary injury cascades. As a consequence of cell lysis, an increase in extracellular Ca(2+) results in additional neuronal loss by inducing apoptosis. Thus, hydrogels that reduce extracellular Ca(2+) concentration may reduce secondary injury severity. The goal of this study was to develop composite hydrogels consisting of alginate, chitosan, and genipin that interact with extracellular Ca(2+) to enable in situ gelation while maintaining an elastic modulus similar to native spinal cord (∼1000 Pa). It was hypothesized that incorporation of genipin and chitosan would regulate hydrogel electrostatic characteristics and influence hydrogel porosity, degradation, and astrocyte behavior. Hydrogel composition was varied to create hydrogels with statistically similar mechanical properties (∼1000 Pa) that demonstrated tunable charge characteristics (6-fold range in free amine concentration) and degradation rate (complete degradation between 7 and 28 days; some blends persist after 28 days). Hydrogels demonstrate high sensitivity to Ca(2+) concentration, as a 1 mM change during fabrication induced a significant change in elastic modulus. Additionally, hydrogels incubated in a Ca(2+)-containing solution exhibited an increased linear viscoelastic limit (LVE) and an increased elastic modulus above the LVE limit in a time dependent manner. An extension of the LVE limit implies a change in hydrogel cross-linking structure. Attachment assays demonstrated that addition of chitosan/genipin to alginate hydrogels induced up to a 4-fold increase in the number of attached astrocytes and facilitated astrocyte clustering on the hydrogel surface in a composition dependent manner. Furthermore, Western blots demonstrated tunable glial fibrillary acid protein (GFAP) expression in astrocytes cultured on hydrogel blends, with some hydrogel compositions demonstrating no significant increase in GFAP expression compared to astrocytes cultured on glass. Thus, alginate/chitosan/genipin hydrogel composites show promise as scaffolds that regulate astrocyte behavior and for the prevention of Ca(2+)-related secondary neuron damage during acute SCI.

Figure 1

Sensitivity of hydrogel elastic modulus to changes in Ca²⁺ concentration and hydrogel composition. A-B) Strain sweeps demonstrating mM level sensitivity to changes in Ca²⁺ concentration for (A) 0.25% or (B) 0.5% alginate hydrogels. C-D) Gelation time tests demonstrating gelation kinetics for alginate or composite hydrogels with a base of (C) 0.25% or (D) 0.5% alginate. E) Comparison of elastic modulus of all hydrogels 30 minutes after beginning of the gelation time tests. n = 3, mean ± standard deviation. (* denotes statistically significance differences between all groups.)

Figure 2

Changes in rheological behavior of hydrogels following in situ gelation modeling with a normal (1.8 mM) Ca²⁺ concentration. Strain sweeps demonstrating the effect of incubation in Ca²⁺ containing media on (A) A25/C0/G0/Ca22, (B) A25/C125/G1/Ca23, (C) A25/C25/G05/Ca18, (D) A5/C0/G0/Ca22, (E) A5/C125/G1/Ca24, and (F) A5/C25/G01/Ca20 hydrogels. n = 3, mean ± standard deviation. (* denotes significance between day 0 and day 2. + denotes significance between day 0 and day 5. # denotes significance between day 2 and day 5.)

Figure 3

Comparison of the change in ultimate elastic modulus following in situ gelation modeling for low and high calcium concentrations. Time tests demonstrating the effect of incubation in media containing low (normal) and high (elevated) Ca²⁺ concentrations for (A) A5/C0/G0/Ca22, (B) A5/C125/G1/Ca24, and (C) A5/C25/G01/Ca20 hydrogels. n = 3, mean ± standard deviation. (Bars with the same letters represent groups in which no statistical differences were observed, while bars with different letters are statistically different from one another.)

Figure 4

Ninhydrin Assay. The number of free amine groups within composite hydrogels is composition dependent. An increase in chitosan concentration and decrease in genipin concentration leads to an increase in the number of free amine groups. n = 3, mean ± standard deviations (* denotes significance between connected groups. # denotes significance between all groups.)

Figure 5

Images (left) and scanning electron micrographs (SEM; right) of alginate and composite hydrogels. A) A25/C0/G0/Ca22, B) A5/C0/G0/Ca22, C)A25/C125/G1/Ca23, D) A5/C125/G1/Ca24, E) A25/C25/G05/Ca18, and F) A5/C25/G01/Ca20. For SEM micrographs: magnification −300X, scale bar −100 μm.

Figure 6

Astrocyte interaction within different hydrogel blends. A-C) Schematics detailing the proposed internal cross-linking nature of hydrogels and cellular interaction for A) alginate hydrogels, B) composite hydrogels with low chitosan and high genipin concentrations, and C) composite hydrogels with high chitosan and low genipin concentration. D-I) Representative fluorescent images of astrocytes attached to different hydrogel blends: D,G) A25/C0/G0/Ca22, E,H) A25/C125/G1/Ca23, and F,I) A25/C25/G05/Ca18 (D-F: 10X Magnification, scale bar −300 μm G-I: 40X Magnification, scale bar −100 μm. Green – Calcein AM, Blue – Hoechst 33342. J-L) Analysis performed on 10X fluorescence images detailing J) the number of cells per sample, K) the number of clusters per sample, and L) the number of cells per cluster per sample for each hydrogel blend. n = 3, mean ± standard deviation (Bars with the same letters represent groups in which no statistical differences were observed, while bars with different letters are statistically different from one another.)

Figure 7

GFAP expression in astrocytes cultured on different hydrogels. A) Representative Western blots showing the GFAP and α-tubulin expression in astrocytes cultured on poly-d-lysine coated glass or hydrogels with different polymer compositions. B) Quantitative analysis performed on Western blots demonstrating the relative GFAP expression in astrocytes cultured on different surfaces. n = 3, mean ± standard deviation. (Bars with the same letters represent groups in which no statistical differences were observed, while bars with different letters are statistically different from one another.)