Construction And Evaluation Of In Situ Hydrogels For Osteoarthritis Based On RONS Clearance

Osteoarthritis（OA）is a common degenerative joint disease characterized by bone erosion,synovial inflammation,and cartilage destruction.The continuous production of large amounts of reactive oxygen and nitrogen species（RONS）and inflammatory factors in the joint cavity are major factors in the worsening inflammation.In this project,an in situ multifunctional composite thermo-sensitive hydrogel for injection was designed.It is designed to treat OA and repair cartilage by slow release of hyaluronic acid（HA）,platelet-rich plasma（PRP）and copper nanodots（Cu NDs）.In brief,HPP@Cu composite thermo-sensitive hydrogel in solution form was injected into the OA inflammatory site.Due to the thermo-sensitive property of Poloxamer 407（P407）,the composite gel was rapidly phase-transformed into a gel in vivo and the contents were slowly released for therapeutic effect.HA provides cartilage protection and lubrication to the joint.PRP promotes cartilage repair and proliferative effects.Cu NDs can efficiently remove RONS from the joint cavity and reverse the pro-inflammatory phenotype of macrophages.The HPP@Cu composite thermo-sensitive hydrogel combined the advantages of each of these components to retard OA progression.Cu NDs were prepared by reducing anhydrous copper sulfate by hydrothermal reduction method.The morphology,particle size and potential of Cu NDs were characterized by transmission electron microscopy（TEM）and dynamic light scattering（DLS）.The results showed that the Cu NDs have an ultra-small uniform size of 5.37±0.92nm and a zeta potential of-10.63±1.37 m V.The nanoparticles were structurally characterized by Fourier infrared spectroscopy（FTIR）,ultraviolet-visible spectroscopy（UV-vis）and X-ray electron diffraction（XRD）,and the results showed that the nanoparticles were composed of Cu NDs wrapped by PVP.The results of stability test showed that Cu NDs were stable in water,PBS,serum and complete medium.The efficient radical scavenging ability of Cu NDs against H₂O₂,NO?,?O₂^-,PTIO,NO?,ABTS?⁺,DPPH was investigated by ultraviolet colorimetry,respectively.Subsequently,HP thermo-sensitive gel matrices were prepared by mixing P407 and HA by cold method.The prescription of the composite thermo-sensitive hydrogel was optimized by gelation temperature,viscosity,and gelation time.The final composite thermo-sensitive hydrogel HPP@Cu was obtained by adding PRP,and Cu NDs to the HP thermo-sensitive gel by a simple physical mixing method.A sparse porous structure was observed inside the HPP@Cu hydrogel by scanning electron microscopy（SEM）.In vitro lysis experiments of the gels observed that the HPP@Cu hydrogels were slowly lysed in vitro and the lysis was nearly complete after 8 days.In vitro release experiments observed that Cu NDs,HA and PRP-derived TGF-β1 could be released slowly in HPP@Cu hydrogels.In addition,dissolution was found to be the main factor determining the release rate of Cu NDs.The RONS removal performance of HPP@Cu composite thermo-sensitive hydrogels was also investigated by colorimetric method.The effects of Cu NDs and HPP@Cu composite thermo-sensitive hydrogels on the cell viability of chondrocytes,RAW264.7,and L929 were studied by MTT assays.The results showed that Cu NDs（0-50 ng/m L）and HPP@Cu hydrogel（50 ng/m L）had almost no effect on the viability of the above cells.The proliferative effect of HPP@Cu hydrogel on chondrocytes was verified by Calcein-AM staining.The results showed that the number of chondrocytes was significantly increased in the composite thermo-sensitive hydrogel group with the addition of PRP compared to the hydrogel group without PRP.The in vitro ROS and RNS scavenging activities of HPP@Cu hydrogels were examined using the DCFH-DA probe and BBoxi Probe^?O52 probe,respectively.The results showed that the HPP composite thermo-sensitive hydrogel incorporating Cu NDs had an efficient scavenging effect on RONS in chondrocytes and RAW264.7.The protective effect of Cu NDs against H₂O₂-induced chondrocyte apoptosis was investigated by MTT assay and Annexin V-FITC/PI apoptosis assay.The results showed that Cu NDs significantly reduced the apoptosis rate of chondrocytes because they scavenged RONS from the cells and reduced the level of oxidative stress.The phenotypic conversion ability of HPP@Cu composite hydrogels on macrophages was subsequently assessed by immunofluorescence experiments.The fluorescence intensity of surface markers（CD206and i NOS）of macrophages showed that HPP@Cu hydrogel was able to promote the anti-inflammatory phenotype switch of macrophages,which was from M1 phenotype to M2 phenotype.The therapeutic effects of HPP@Cu composite thermo-sensitive gel on monosodium iodoacetate（MIA）-induced OA model in rats were investigated.The results of in vivo experiments showed that HPP@Cu complex thermo-sensitive hydrogel was able to clear RONS from joint tissues and reduce the level of inflammatory factors in joint sites.Through the observation of joint specimens and their pathological sections,the results showed that HPP@Cu hydrogel was able to protect the structural integrity of cartilage tissue and the cartilage layer in the joint area was not damaged.In addition,immunofluorescence experiments showed that HPP@Cu hydrogel could promote the conversion of synovial macrophages from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype and exert anti-inflammatory effects.In conclusion,the multifunctional HPP@Cu composite thermo-sensitive hydrogel improved the inflammatory microenvironment at OA sites and repaired cartilage by scavenging RONS and reprogramming the macrophage phenotype.This study provides a reliable reference for the application of injectable hydrogels in inflammatory diseases associated with oxidative stress.