Experimental Study On The Therapeutic Effects Of Polydopamine Nanoparticles In Temporomandibular Joint Osteoarthritis

Objectives:Because of the obscure etiology of temporomandibular joint osteoarthritis（TMJ-OA）and unsatisfactory treatment outcomes,new effective and minimally invasive therapies are urgently needed.Oxidative stress elicited by excessive reactive oxygen species（ROS）accumulation has historically been considered a potential trigger of TMJ-OA.Polydopamine（PDA）,an emerging versatile biopolymer produced by self-polymerization of dopamine,has attracted considerable attention in biomedical applications by virtue of its excellent biocompatibility and intriguing ROS-scavenging capacity.Herein,we study the antioxidative and anti-inflammatory capacity of PDA to open a new avenue for TMJ-OA treatment.Materials and methods:1.Synthesis of PDA:PDA nanoparticles（NPs）were synthesized in the ethanol solution（p H=8.5）at 37℃for 24h and their physicochemical properties were characterized by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,dynamic light scattering（DLS）and Fourier transform infrared（FT-IR）spectral analysis.2.Biological evaluation of PDA NPs in chondrocytes:CCK-8 assays and live/dead cell staining were performed to assess the biocompatibility of PDA NPs.The scavenging capacities of PDA NPs upon mitochondrial/intracellular/extracellular ROS were investigated.The effects of PDA on oxidative phosphorylation（OXPHOS）of chondrocytes were studied by extracellular flow analyzer,thereby revealing the mechanism underlying the excellent ROS-scavenging capacity of PDA.Changes of mitochondrial membrane potential（ΔΨm）of LPS-induced chondrocytes were studied to assess the effect of PDA on mitochondrial functions.Polymerase chain reaction（PCR）and Western blot analyses were performed to evaluate the inflammatory cytokine levels in LPS-induced chondrocytes while live/dead cell staining and flow cytometry were performed to explore the anti-inflammatory effects of PDA in vitro.3.Biological evaluation of PDA NPs in the rat TMJ-OA model:Firstly,we established a monosodium iodoacetate（MIA）-induced TMJ-OA model.PDA NPs of different concentrations(40μg m L^-1,80μg m L^-1,and 120μg m L^-1)were intraarticularly injected in TMJ-OA rats.The attenuation of TMJ-OA symptoms as well as histological changes were assessed by weight measurement,Micro-CT scanning,determination of tissue ROS level,and histological staining.The biosafety of PDA was revealed by hematoxylin and eosin（H&E）staining of the main organs of TMJ-OA rats.Results:1.PDA NPs synthesized in this study were uniform and monodisperse nanospheres with an average diameter of 117.7±8.3nm and an average Zeta potential of-39.8±4.2 m V.The FI-TR spectroscopy peaks at 1606 cm^-1,1415 cm^-1,and 1288 cm^-1 were attributed to the C=O,C=C,and C-O bonds,indicating the successful formation of PDA.2.The excellent biocompatibility of PDA was confirmed in chondrocytes.After incubation with the 200μg m L^-1 PDA NPs for 72h,no obvious inhibition of the proliferative activity of chondrocytes was observed.Furthermore,PDA NPs exhibited excellent ROS scavenging ability and inhibitory effect on lipopolysaccharide（LPS）-induced chondrocyte apoptosis in vitro.Moreover,PDA NPs could increase the OXPHOS rate of mitochondria in chondrocytes under inflammatory stimulation by improving the respiration rate at Complex II,III,and IV,thereby reducing the production of intracellular ROS and improving the efficiency of energy metabolism.The ATP production deficiency as well as the decline ofΔΨm was markedly alleviated after incubation with PDA NPs.In addition,120μg m L^-1 PDA NPs showed the most significant inhibition of LPS-induced inflammation in chondrocytes,thereby used in the in vivo study.3.Intraarticular injection of PDA NPs in the TMJ-OA rat model showed an accelerated weight gaining,indicating alleviated TMJ-OA symptoms.Moreover,injection of PDA NPs into articular capsule in the TMJ-OA rat model not only showed a satisfactory general biosafety but also reduce the levels of ROS and matrix metalloproteinase-13（MMP-13）in condyle,inhibit apoptosis and osteoclast formation,which is beneficial to alleviate the cartilage degeneration and subchondral bone destruction in TMJ-OA.Conclusions:In summary,we successfully synthesized PDA NPs with good biocompatibility,excellent ROS-scavenging ability,and anti-inflammatory capacity.We revealed that PDA NPs may reduce ROS production by increasing the OXPHOS efficiency in Complex II,III,and IV,thereby improving the energy metabolism in LPS-induced chondrocytes.The results of animal study showed that PDA NPs can improve the condylar cartilage degeneration and subchondral bone destruction of TMJ-OA rats,which not only provides a new idea for the clinical treatment of TMJ-OA,but also has important clinical value and scientific significance.