Biocompatible Amino-modified Tantalum Nanoparticles Regulate Oxidative Stress In Osteoarthritis

Background:The most prevalent form of degenerative joint disease is osteoarthritis（OA）,which is associated with multiple factors such as genes,metabolic disorders,diet and rest,obesity,long-term physical labor and previous joint injury.The cardinal feature of this disease is the dysfunction of articular cartilage,which can lead to obvious joint pain,joint deformation and eventually limited mobility of patients in the terminal stage.More and more researches revealed that oxidative stress is key factor in the progression of OA in recent years.Therefore,reducing oxidative stress and reactive oxygen species（ROS）production in joints is a reasonable strategy for the treatment of OA.However,fluctuations in the physical and chemical microenvironment of osteoarthritis lead to instability in the properties of existing antioxidants.In addition,poor biocompatibility and short joint retention time also seriously hinder the clinical application of antioxidant therapy.In order to overcome the above deficiencies,tantalum,which is commonly used in clinical practice,was selected as the research target and modified to prepare positively charged tantalum nanoparticles（Ta-NH₂NPs）with high biocompatibility and continuous catalase-like activity in joint cavity.Catalase（CAT）was used as a control to further explore the potential of Ta-NH₂ NPs as an effective ROS scavenger and its therapeutic effect on OA;PurposesIn this study,we explored the potential of Ta-NH₂ NPs to decompose H₂O₂,effects on the phenotype chondrocytes of rat OA,biocompatibility in vivo and in vitro in rats and the therapeutic effect of OA in rat;MethodPart 1:Preparation,characterization,H₂O₂ scavenging activity and in vitro biocompatibility of Ta-NH₂ NPs1.Preparation of Ta-NH₂ NPsAfter the NPs of large particles was removed by centrifuge,we determined the concentration of the collection solution by oven drying method.Then the concentration of NPs solution was diluted to 1 mg/m L by ethanol.Appropriate 3-amino-propyl-3-methoxylsilane was added to the solution and stirred at 70℃for 3 h.After centrifugation,Ta-NH₂ NPs was obtained by finally washing;2.Characterization of Ta-NH₂ NPsThe samples were prepared,sample morphology,chemical element analysis,hydrodynamic sizes,particle dispersity index（PDI）,X-ray diffraction（XRD）,zeta-potential,Fourier transforms infrared（FT-IR）spectra and X-ray photoelectron spectroscopy（XPS）were characterized by some specific instruments next;3.In vitro H₂O₂ scavenging and stability detection under different environments of Ta-NH₂ NPsDifferent concentrations of Ta-NH₂ NPs and 1mM H₂O₂ were mixed and cocultured at37℃for 1 h.The decomposition concentration and efficiency were measured after the reaction.After that,the selected concentration of Ta-NH₂ NPs（100μg/m L）and 1 m M H₂O₂were fully mixed and reacted for 1h at different temperature and p H,and the decomposition efficiency of Ta-NH₂ NPs was measured follow;4.Rat chondrocyte extraction and cultureFemale SD rats were killed by CO₂ asphyxia and the knee cartilage was separated.The cartilage was broken up with ophthalmic scissors and digested by type II collagenase.After filtering out cartilage residue,primary chondrocytes were obtained.The acquired chondrocytes were grown in a 5%CO₂ incubator at 37°C with complete Dulbecco’s modified Eagle’s media（DMEM）.The experimental cells were selected as P2 generation rat chondrocytes;5.In vitro biocompatibility test of Ta-NH₂ NPsRat chondrocytes were stimulated with Ta-NH₂ NPs at different concentrations and the cytotoxicity of Ta-NH₂ NPs was determined by the CCK-8 assay.For purpose of further proving the reliability,cell viability was assessed by morphology of Rat chondrocytes,Calcein-AM/PI staining and Krystal violet staining.Rat chondrocytes were then stimulated by specific concentration（100μg/m L）,Western-Blot was used to detect the phenotype change.Alcian blue staining,and immunofluorescence staining to further test then main chondrocyte phenotypes;Part 2:Effects of Ta-NH₂ NPs on the phenotype of rat OA chondrocytes in vitro1.H₂O₂ induced cell oxidative stress in vitroChondrocytes were pre-treated with Ta-NH₂ NPs（100μg/m L）for 2 h.400μM H₂O₂ was then added into and cells were incubated for another 24 h;2.Treatment of rat OA chondrocytesAfter completing the above processing,the following tests（Immunofluorescence and Western-Blot）were tested the expression of ROS,iNOS,COL-II,ACAN,COL-I,ADAMTS-5,RUN-2 and MMP-13;Part 3:In vivo biocompatibility evaluation and in vivo therapeutic effect of Ta-NH₂ NPs1.Fluorescent dye labelingTa-NH₂ NPs and CAT were labeled with near-infrared dye CY5.5;2.The retention time and biodistribution of antioxidant in the articular cavity of ratsMale SD rats were intra-articularly injected with Ta-NH₂ NPs-CY5.5,CAT-CY5.5 and CY5.5.The fluorescence images of rat knees were captured at 1,3,7,14 and 28d.The knee joint,femur condyles,and organs including heart,brain,lung,kidney,liver and spleen)were harvested for ex vivo NIR imaging.The harvested rat femur condyles were then fixed embedded,TEM was used to further test the element distribution in the femoral condylar cartilage layer;3.In vivo toxicity evaluation of Ta-NH₂ NPsBlood samples were harvested at day 1 and 28 after intra-articular injection of Ta-NH₂NPs.Liver function test（Aspartate aminotransferase and alanine aminotransferase）,kidney function test（blood urea nitrogen and creatinine）and complete blood count was done to evaluate the in vivo toxicity of Ta-NH₂ NPs.Furthermore,the harvested major organs were stained by hematoxylin and eosin（H&E）;4.Intra-articular injection MIA was used for inducing OA models.The next experiment will be conducted 2 weeks after injection.After models completed,Ta-NH₂ NPs and CAT were injected into knee joint.Rats were sacrificed after treatment with Ta-NH₂ NPs or CAT for 4 or 8 weeks.The knee joints were collected and fixed in paraformaldehyde;5.Micro CT scan of rat knee jointEight weeks after the antioxidants injection,the knee joint samples were obtained and imaged by Micro CT scan;6.Histological section stainingKnee joint samples of rats were obtained after antioxidants injection 4 weeks or 8 weeks.Then,the samples were fixed-decalcified and embedded-sectioned.Samples then were stained by iNOS,HE and Safranin O-Fast Green;ResultPart 1:Preparation,characterization,H₂O₂ scavenging activity and in vitro biocompatibility of Ta-NH₂ NPs1.Ta-NH₂ NPs were successfully prepared after a series of steps.Through various aspects of the material characterization,it was further confirmed that Ta NPs had been successfully modified by amino groups.2.Ta-NH₂ NPs exhibited ROS scavenging activity in a concentration-dependent manner.Approximately 35%of the H₂O₂ was decomposed by 50μg/m L Ta-NH₂ NPs,and almost 40%H₂O₂ could be scavenged in the concentration 100μg/m L with excellent p H and temperature stabilities;3.The CCK-8 assay results showed no significant cytotoxicity at test concentration after24 h and 48 h co-culture.But slightly decreased chondrocyte viability was noticed when concentration reached 200μg/m L.Chondrocytes cultured with Ta-NH₂ NPs did not manifest obvious morphology change.Crystal violet staining,alcian blue staining,and immunofluorescent staining of COL-II and ACAN results indicated that Ta-NPs treatment did not affect the deposition of cartilaginous ECM.4.Western blot results showed that there were significant decreases in protein levels of COL-II,SOX-9,RUNX-2,and ADAMTS-5.Subsequent quantitative analysis further supported these results;Part 2:Effect of Ta-NH₂ NPs on the phenotype of rat OA chondrocytes in vitro1.DCFH-DA staining indicated significant increase in intra-cellular ROS level in H₂O₂treated group,which was inhibited by either Ta-NH₂ NPs or CAT.Our data showed remarkably high expression of iNOS after H₂O₂ challenge.In contrast,the expression of iNOS was reversed after adding Ta-NH₂ NPs,but slightly increased after CAT treatment.In addition,live and dead staining data indicated Ta-NH₂ NPs or CAT pre-treatment protected chondrocytes viability via inhibiting intra-cellular ROS production;2.The levels of ACAN and COL-II expression were obviously decreased after H₂O₂stimulation compared with the normal group.Compared with the H₂O₂ group,the expression levels of COL-II and ACAN in rat chondrocytes were remarkably increased after adding antioxidants,and the effect of Ta-NH₂ NPs was more obvious than that of CAT;3.The expression levels of COL-I,ADAMTS-5,RUNX-2 and MMP-13 in rat OA chondrocytes were also significantly increased with the same treatment.Compared with the no antioxidant treatment group,the expression levels of COL-I,ADAMTS-5,RUNX-2 and MMP-13 in rat chondrocytes were significantly decreased after adding antioxidants,and the effect of Ta-NH₂ NPs was more obvious than that of CAT;Part 3:In vivo biocompatibility evaluation and in vivo therapeutic effect of Ta-NH₂ NPs1.After intra-articular injection of fluorochrome CY5.5 labelled Ta-NH₂ NPs and CAT,in vivo fluorescence imaging indicated that gradually decreased concentration of both antioxidants at day 1,3,7,14 and 28.The CY5.5 dye alone showed rapid decrease in fluorescence intensity 1 h post injection.Compared with CAT group（at day 3）,significantly longer joint retention（at day 28）of Ta-NH₂ NPs was noticed;2.The results of in vivo fluorescence imaging showed that fluorescence could be continuously detected in the femoral condyle of the rat knee joint in the CY5.5 labeled Ta-NH₂ NPs group,but not in the CY5.5 labeled CAT group.The elemental analysis of the cartilage layer of the rat knee joint showed that there was a large amount of Ta accumulation in the cartilage layer;3.At the macroscopic level,Ta-NH₂ NPs were mainly accumulated in the liver,spleen and kidney.Among them,the fluorescence intensity in spleen,liver and kidney reached the peak at day 1,3 and 7 post injection and decayed by day 3,7 and 14 respectively;4.No hemorrhage,atrophy,or necrosis was found in the analyzed organs.Liver and kidney function tests and blood biochemistry tests showed that some indicators fluctuated significantly after injection of CY5.5 labeled Ta-NH₂ NPs,but the values were still in the normal range;5.The micro-CT scanning indicated obvious bone defects in the patella and femoral condyle,and alternation in subchondral bone structure in vehicle group.The subchondral bone structure was significantly improved by Ta-NH₂ NPs or CAT injection;6.The iNOS expression in articular cartilage was significantly rising,and the expression reached the peak at the 8th week after MIA injection.Antioxidant treatment decreased the expression of iNOS,particularly in Ta-NH₂ NPs treated group.Ta-NH₂ NPs,but not CAT showed prolonged inhibition of iNOS expression at the 8th week after MIA injection.The expression of iNOS in synovial tissue was similar to that in cartilage,while the iNOS expression of all groups in subchondral bone are same.7.MIA injection induced synovial inflammation.In vehicle group,obvious hyperplasia and inflammatory cell infiltration were seen in synovium at 4 weeks post intra-articular injection.Vessel hyperplasia was also noticed at 8 weeks.The inflammation of the synovium was alleviated after both anti-oxidative treatments.Notably,synovitis score indicated synovial inflammation in the Ta-NH₂ NPs group was significantly lower than that in the CAT group at8 weeks post injection;8.The vehicle group presented typical OA features such as surface irregularity,decreased expression of glycosaminoglycans,and cartilage defects.These cartilage damages were alleviated after antioxidant treatment.The Ta-NH₂ NPs treatment presented sustained remission at week 8,while CAT treatment did not exert its antioxidant function at this time point.Compared with vehicle group,both antioxidants attenuated the OARSI score in femur,and significantly decreased the OARSI score in tibia.Ta-NH₂ NPs exhibited excellent long-term therapeutic effect as evidenced by more homogeneous glycosaminoglyc and cell arrangement;Conclusion1.The results showed that our designed Ta-NH₂ NPs had good biocompatibility and stability,and protected viability and hyaline-like phenotype in chondrocyte under oxidative stress;2.Intra-articular injection of Ta-NH₂ NPs can effectively alleviate the level of synovial inflammation and delay the progression of OA in rats induced by MIA;3.Ta-NH₂ NPs showed sustained retention in the joint cavity,particularly in articular cartilage.Ta-NH₂ NPs exhibited long-term anti-oxidant stress and therapeutic effects in MIA-induced OA model;...