Thermosensitive Chitosan/Gelatin/Glycerol Phosphate Hydrogel as a Sustained Release System of Ferulic Acid for Nucleus Pulposus Regeneration
Date Issued
2012
Date
2012
Author(s)
Cheng, Yung-Hsin
Abstract
Disc degeneration is strongly associated with back pain and herniation that increase the costs of health care. The degeneration of intervertebral disc (IVD) could be divided into 5 stages. In the first and second stages, there are no significant symptoms but could be traced by magnetic resonance imaging or computed tomography-scan. Generally, no aggressive treatment would be processed in the clinics. Recent studies indicated that overproduction of reactive oxygen species (ROS) may accelerate the degenerative process of IVD and associate with apoptosis of nucleus pulposus (NP) cells and degradation of extracellular matrix. Ferulic acid (FA) is an excellent antioxidant and relatively stable in air. FA has been proven to have ability to prevent ROS-induced diseases. The object of the study was aimed to evaluate the possible therapeutic effect of FA on hydrogen peroxide (H2O2)-induced oxidative stress NP cells and the feasibility of use the thermosensitive chitosan/gelatin/glycerophosphate (C/G/GP) hydrogel as a sustained release system of FA for early treatment in IVD degeneration.
In the study, NP cells were harvested from the IVD of New Zealand rabbits. The results showed that 500 μM of FA might be the threshold to treat NP cells without cytotoxicity. Post-treatment of FA on H2O2-induced oxidative stress NP cells significantly up regulated the expression of aggrecan, type II collagen and BMP-7 and down regulated the expression of MMP-3 in mRNA level. Post-treatment of FA on H2O2-induced oxidative stress NP cells could restore the production of sulfated glycosaminoglycans (GAGs) and inhibit the apoptosis caused by H2O2. The results showed that the release of FA from C/G/GP hydrogel could decrease the H2O2-induced oxidative stress. Post-treatment of FA-incorporated C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed up-regulation of aggrecan and type II collagen and down-regulation of MMP-3 in mRNA level. The results of sulfated GAGs to DNA ratio and alcian blue staining revealed that the GAGs production of H2O2-induced oxidative stress NP cells could reach to normal level. The results of caspase-3 activity and TUNEL staining indicated that FA-incorporated C/G/GP hydrogel decreased the apoptosis of H2O2-induced oxidative stress NP cells. The results showed that FA was successfully immobilized on C/G/GP hydrogel by N-(3-dimethylaminopropyl)-N''-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinking method. The gelation temperature of the FA-immobilized C/G/GP hydrogel was 31.80 degree celsius under neutral pH. Post-treatment of FA-immobilized C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed down-regulation of MMP-3 and up-regulation aggrecan and type II collagen in mRNA level. The sulfated GAGs production of H2O2-induced oxidative stress NP cells could be increased to the normal level in the post-treatment of FA-immobilized C/G/GP hydrogel group. The results of caspase-3 activity and TUNEL staining showed that the apoptosis of H2O2-induced oxidative stress NP cells could be inhibited by post-treatment of FA-immobilized C/G/GP hydrogel.
From the results of the study, FA could be used as a therapeutic molecule for NP regeneration and FA-incorporated C/G/GP hydrogel might be potentially applied as a long-term release system. The immobilization of FA on C/G/GP hydrogel could significantly prolong the release period of FA. These results suggest that combination of FA and thermosensitive C/G/GP hydrogel can treat NP cells from the damage caused by oxidative stress and may apply in minimally invasive surgery for NP regeneration in the future.
Subjects
nucleus pulposus
ferulic acid
oxidative stress
thermosensitive hydrogel
antioxidant
Type
thesis
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