Application Of ROS-responsive Magnesium-containing Microspheres In The Treatment Of Intervertebral Disc Degeneration

Objective:To evaluate the in vitro and in vivo biocompatibility of ROS-responsive magnesium-containing microspheres（Mg@PBE/PLGA MSs）,and evaluate their effects on intervertebral disc degeneration.Methods:（1）Different concentrations of Mg@PBE/PLGA MSs were incubated with RAW264.7 cells,and the effects of different concentrations of Mg@PBE/PLGA MSs on cell viability were detected by CCK-8 experiment.The microspheres were incubated with rat erythrocytes.The erythrocyte toxicity and in vitro biocompatibility of Mg@PBE/PLGA MSs were evaluated by hemolysis experiment.（2）The microspheres were injected into the intervertebral disc of rats.The important organs of rats（heart,liver,spleen,lung and kidney）obtained after injecting the microspheres into the intervertebral disc were paraffin sectioned and HE stained to evaluate the biocompatibility of Mg@PBE/PLGA MSs in vivo.（3）A rat model of caudal intervertebral disc degeneration induced by acupuncture was established,and Mg@PBE/PLGA MSs were injected into the intervertebral disc.Before and 4 weeks after puncture,the changes of intervertebral space were observed by electronic computed tomography（CT）,and the changes of disc height index（DHI）were analyzed.The changes of signal intensity in the nucleus pulposus area,the structures of the intervertebral discs were detected by 1.5 T magnetic resonance imaging（MRI）.Pfirrmann score was performed according to the MRI results.Besides,the histomorphological changes of intervertebral discs were observed by histopathological staining（including HE staining,Masson staining and Safranin O staining）.Immunohistochemical staining（IHC）was used to observe the changes of proteoglycan content,matrix degrading enzymes,oxidative stress and inflammation level in intervertebral disc.The above methods were used to comprehensively evaluate the degree of intervertebral disc degeneration in rats and the effect of Mg@PBE/PLGA MSs on intervertebral disc degeneration in vivo.Results:（1）The results of CCK-8 experiment showed that the microspheres had no significant effect on cell viability in the concentration range of less than 2mg/m L,and had good biocompatibility.The hemolysis test results showed that the microspheres had no obvious erythrocyte toxicity and low hemolysis rate,which proved the safety of the microspheres in vitro.（2）The HE staining results of rat important organs（heart,liver,spleen,lung and kidney）obtained after intradiscal injection of microspheres showed that the microspheres had good biocompatibility and no obvious tissue toxicity in vivo.（3）In the in vivo study,after 4 weeks of puncture,CT examination of rats in the model group showed that the intervertebral space became narrow,the intervertebral disc height and DHI decreased significantly.MRI showed decreased signal intensity in the nucleus pulposus area and increased Pfirrmann score.The results of HE staining,Masson staining and Safranin O staining showed significant decrease of the content of nucleus pulposus and extracellular matrix,fiber proliferation,and annulus fibrosus structure disorder.IHC results showed that the content of proteoglycan in intervertebral disc decreased significantly,while the levels of matrix degrading enzymes,oxidative stress and inflammation increased significantly.The above results proved that the intervertebral disc underwent serious degeneration.However,in the experimental group,the injection of Mg@PBE/PLGA MSs could significantly alleviate the above changes.Conclusions:Mg@PBE/PLGA MSs had good biocompatibility in vivo and in vitro.In vivo experiments showed that the microspheres had great antioxidant and anti-inflammatory effects,could effectively prevent intervertebral disc degeneration,and provided a new strategy for the treatment of intervertebral disc degeneration.