Facile fabrication of an erythropoietin-alginate/chitosan hydrogel and evaluation of its local therapeutic effects on spinal cord injury in rats

Abstract

Background and objective: Spinal cord injury (SCI) is a major disabling disorder for which no effective treatment has yet been found. Regenerative incapability of neuronal cells as well as the secondary mechanisms of injury are the major reasons behind this clinical frustration. Thus, here we fabricated an erythropoietin-chitosan/alginate (EPO-CH/AL) hydrogel and investigated its local therapeutic effects on the apoptotic and inflammatory indices of SCI secondary injury.

Methods: EPO-CH/AL hydrogels were fabricated by the ionic gelation method, and they were characterized using SEM and FTIR. In vitro drug release profile of EPO-CH/AL hydrogels was evaluated by UV-vis spectroscopy. Experimental SCI was inflicted in rats which were then treated with CH/AL hydrogels containing different doses of EPO (1000, 5000 and 10,000 IU/kg). The relative expression of Bax and Bcl2 (apoptosis index) and active and inactive forms of NF-κB (inflammation index) were assessed using western blot. Total serum levels of TNF-α were also assessed with ELISA, and histopathological and immunohistochemistry studies were carried out to check the overall changes in the injured tissues.

Results: In vitro drug release test indicated that the EPO-CH/AL hydrogels had a sustained- and controlled-release profile for EPO under these conditions. All the fabricated hydrogels dramatically reduced the elevated inflammation and apoptosis indices of the SCI-inflicted rats (p ≤ 0.05). Nevertheless, only EPO-CH/AL hydrogel (1000 IU/kg EPO) significantly improved the tissue repair and histopathological appearance of the spinal cord at the sites of injury.

Conclusion: Based on our findings, EPO-CH/AL hydrogel (1000 IU/kg EPO) can effectively improve experimental SCI in rats via inhibiting apoptosis and inflammation. Further studies are warranted to elucidate the contributing role of the scaffold in the observed effects.

Keywords: Alginate; Apoptosis; Chitosan; Erythropoietin; Inflammation; Spinal cord injury.

Fig. 1

FTIR spectra of the carboxymethylcellulose, sodium alginate, chitosan, and the final formulation

Fig. 2

Scanning electron microscopy (SEM) micrograph of EPO-CH/AL

Fig. 3

Release profile of A EPO1-CH/AL, B EPO5-CH/AL and C EPO10-CH/AL hydrogels

Fig. 4

The viability of U373-MG cells after 24, 48 and 72 h

Fig. 5

The expression of NF-κB, Bax and Bcl-2 in different groups defined by western blot (β-actin was also used as an internal control). p- NF-κB and t- NF-κB represent the phosphorylated and the truncated forms of NF-κB, respectively. p-NF-κB /t-p-NF-κB ratio was used as the inflammation index. Bax/Bcl-2 was deemed as the apoptosis index. ^# and ^* represent significant differences compared to the control and SCI groups, respectively; ^#/* p ≤ 0.05; ^##/** p ≤ 0.01; ^###/*** p ≤ 0.001

Fig. 6

Total serum concentrations of TNF-α in different experimental groups measured by ELISA. The values are presented as pg/mg protein. ^# and ^* represent significant differences compared to the control and SCI groups, respectively; ^#/* p ≤ 0.05; ^##/** p ≤ 0.01; ^###/*** p ≤ 0.001

Fig. 7

Light micrographs of the spinal cord in different experimental groups. Dotted lines represent lesion area. H&E (A-J) and S100 (K–O) staining. # and * represent significant differences compared to the control and SCI groups, respectively; #/* p ≤ 0.05; ##/** p ≤ 0.01; ###/*** p ≤ 0.001