Lovastatin-Loaded Nanoparticles in a Thermosensitive Chitosan Hydrogel as a Sustained Release System for Treating Intervertebral Disc Degeneration: In Vitro Cell Culture Study and In Vivo Evaluation Using a Needle-Puncture Induced Degeneration Model in Rat Caudal Discs.

Abstract

Sustained release of therapeutic factors, such as lovastatin, is theoretically more beneficial than a single injection in promoting anabolic stimulation to nucleus pulposus cells/tissues. This study aimed to develop lovastatin-loaded nanoparticles (LVS-NPs) incorporated into thermosensitive chitosan/gelatin/glycerophosphate (C/G/GP) hydrogel for the treatment of intervertebral disc (IVD) degeneration. LVS-NPs were prepared using an oil-in-water emulsion evaporation method, followed by characterization and drug encapsulation efficiency. LVS-NPs were then incorporated into C/G/GP hydrogel for controlled drug release, with subsequent in vitro and in vivo evaluations. LVS-NPs showed a round shape with a mid-range polydispersity index, which shall benefit lovastatin release. Rheological study showed incorporation of LVS-NPs into hydrogel does not change thermal gelation property. Drug release tests demonstrated that lovastatin was released steadily from the hydrogel containing LVS-NPs over 7 days. The LVS-NPs-containing hydrogel demonstrated good biocompatibility and low cytotoxicity in the in vitro studies including WST-8 assay, LDH assay, TUNEL test and Western blot. Analysis of hydrogels containing LVS-NPs on human nucleus pulposus cells revealed enhanced production of aggrecan and Type II collagen without inducing apoptosis, though higher concentrations or longer treatment durations may be required to achieve effects comparable to those of 5 μM lovastatin. In the animal study, various treatment regimens were applied to a rat caudal disc model mimicking early-stage IVD degeneration. Despite lovastatin or LVS-NPs in hydrogel, significant decreases in disc height index and extensive degeneration observed through MRI and histological staining indicated a complete loss of nucleus pulposus tissue in all hydrogel-treated groups, likely due to mechanical compression from the injected hydrogel within the confined space of the annulus fibrosus, which needs further investigation. LVS-NPs demonstrated sustained release of lovastatin and extended stimulatory effects on human nucleus pulposus cells in vitro. However, further research is needed to optimize the hydrogel volume and its ratio to intradiscal space and increase lovastatin concentration within LVS-NPs to achieve optimal therapeutic outcomes while minimizing mechanical compression to surrounding tissues.