Oxygen Metabolism-balanced Engineered Hydrogel The Regeneration Of The Nucleus Pulposus By Inhibiting Acid-sensitive Complexes

Objective:Acording to the chain pathological changes stimulated by ROS(oxygen free radical)in the environment of interdisc degeneration(IVDD),chitosan(CS)loaded with black phosphorus quantum dots(BPQDs)was grafted onto the surface of GelMA porous microspheres via amide bond to prepare oxygen metabolic equilibrium engineering injectable hydrogel microspheres GM@CS-BP.The composite microspheres can achieve efficient and uniform loading of nanoparticles,continuously release strong reducing BPQDs,neutralize ROS in situ,reduce oxidative stress damage,and improve the intervertebral disc oxygen metabolism microenvironment.Methods:To explore the positive-feedback interaction between oxidative stress and acid-sensitive ion channels in the intervertebral disc degeneration environment of rats,and explore the mechanism of molecular channels in nucleus pulposus cells that activate acid-sensitive channels under oxidative stress.Secondly,chitosan nanoparticles and black phosphorus quantum dots were prepared by ion crosslinking method and liquid phase stripping method,and porous GelMA hydrogel microspheres(GM)were prepared by microfluidic technology.Chitosan-black phosphorus quantum dot nanoparticles(CS-BP)were connected to microspheres by means of amide bond in 1-(3-dimethylamino-propyl)-3-ethylcarbon diamide hydrochloride/n-hydroxy succinimide(EDC/NHS)system to form oxygen metabolically balanced engineered hydrogel microspheres(GM@CS-BP).Chitosan nanoparticles and black phosphorus quantum dots were characterized by transmission electron microscopy(TEM),Raman shift and nanoparticle.The microspheres were characterized by scanning electron microscopy(SEM),infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(EDS),optical microscopy(BM)and inductively coupled plasma atomic emission spectrometry(ICP).After that,the microspheres were co-cultured with rat nucleus pulposus cells(NPCs)of the caudal intervertebral disc,and hydrogen peroxide(H2O2)was used to intervene in the simulated intervertebral disc degeneration environment.The biocompatibility of GM@CS-BP was explored by the live-dead staining kit and CCK-8 kit.DCFH-DA fluorescent probe.ABTS kit and JC-1 kit were used to investigate the antioxidant and anti-apoptotic abilities of composite microspheres.The inhibition of acid-sensitive complex 3(ASIC-3)and its regulatory effect on inflammatory factors expressed in nucleus pulposus cells were investigated by Immunofluorescence staining(Immunofluorescence),western blotting(WB)and real-time quantitative fluorescence PCR(qRT-PCR).Finally,a rat model of caudal vertebra acupuncture was constructed and injected into GM@CS-BP with a micropump syringe to evaluate the in vivo nucleus pulposus repair ability of composite microspheres.X-ray and magnetic resonance imaging(MRI)were used to explore the intervertebral disc image characteristics,and H&E.S-O-Fast Green,immunohistochemical and immunofluorescence staining were used to explore the ability of composite microspheres to promote nucleus pulposus regeneration and regulate inflammation.Results:Black phosphorus quantum dots and chitosan nanoparticles with uniform distribution were prepared,and the physicochemical properties of various CS-BP were investigated.The engineered hydrogel microsphere structure of microsphere-nanoparticlequantum dot was synthesized.The element composition of the composite microsphere was investigated by FTIR and EDS,and the release curve of black phosphorus quantum dot was investigated by ICP.The optimum volume ratio between CS-BP and GM is also investigated.In vitro experiments.CCK-8 experiment and live/dead staining proved that the engineered hydrogel microspheres had good biocompatibility and promoting proliferation of nucleus pulposus cells.DCFH-DA,ABTS and JC-1 experiments demonstrated excellent antioxidant and anti-apoptotic abilities.Western blotting and real-time quantitative PCR showed that the complex microspheres had good regulatory functions on acid-sensitive complex and various inflammatory factors.In vivo experiments.a rat coccygeal puncture model was successfully constructed.Imaging results and tissue section results showed that composite microspheres could not only inhibit acid-sensitive reactivator in the intervertebral disc degeneration environment,but also block the degradation of extracellular matrix(ECM),thus delaying intervertebral disc degeneration and promoting the regeneration of nucleus pulposus.Conclusion:We found that ROS stimulation directly upregulated the expression of the ASIC-3 protein in NP cells and further activated the release of inflammatory factors,such as TNF-α and IL-1β.This not only proves that the imbalance of oxygen metabolism leads to the degradation of the ECM in NP tissue and accelerates IVDD,but also explores the positive feedback between ROS and acid-sensitive complexes.These findings further provide new insights into anti-ROS therapy for IVDD.On this basis,novel oxygen metabolism-balanced engineered hydrogel micro-spheres were constructed to regulate the imbalance of ROS in IVDD precisely by the uniform loading and targeting and sustained release of strong antioxidant BPQDs from CS.The composite microspheres were found to downregulate the expression of the acid-sensing complex in NP cells under high-intensity ROS stimulation;block the activation of downstream inflammatory pathways;and block the vicious cycle be-tween oxidative stress and inflammation to achieve ECM stability in IVDD,restore tissue function,and promote NP regeneration.The oxygen metabolism-balanced engineered hydrogel microspheres constructed in this study therefore represent a new approach for IVDD treatment through a local anti-oxidative stress strategy to regulate the expression of acid-sensing complexes.