Porous Nanocomposite Se@SiO 2 Was Used To Treat Steroid-induced Femoral Head Necrosis By Inhibiting Oxidative Stress

BackgroundSteroid-induced osteonecrosis of the femoral head?SONFH?is one of the most common diseases in orthopedics and is the first in the incidence of Nontraumatic Osteonecrosis of Femoral Head?NONFH?.Because of the widespread use of the glucocorticoids in the clinical,and the irreplaceability of the glucocorticoid therapy,making it has become the primary risk factor for NONFH.At present,hip replacement is the only option for the final stage of treatment of SONFH.SONFH has a high morbidity,causing serious damage to the health of the patient,and creating a heavy family and socio-economic burden.So,find a new treatment method is necessary.Although many hypotheses about the pathogenesis of the disease have been proposed,including lipid metabolism disorders,intravascular coagulation,microcirculation,and increased intraosseous pressure,the exact mechanism of SONFH head remains unclear.By report,oxidative stress disorder is closely related to the development of SONFH.Selenium?Se?is a trace element of the human body,the study found that the selenium?Se?plays an important role in the inhibition of oxidative stress,it has been proved that Se could effectively reduce oxidative stress on DNA damage.In addition,in some special cases,Se deficiency can directly induce oxidative stress and endoplasmic reticulum stress,causing oxidative stress injury.So Se as a good antioxidant has great potential for clinical treatment.However,the application window of the Se element is narrow,seriously restricting its therapeutic effect.With the rapid development of nanotechnology,Nano-Se modified by Nano-technology.It could reduce the risk of toxicity,increase its possible as a drug treatment of disease.The porous nanocomposite Se@SiO₂ used in this study is a new kind of nanomaterials.It is proved that its biocompatibility is good and is the safe and ideal source of Se.It has the ability to control the release,because of the porous structure.And It can be release of the amount of Se slowly for a long time.So that it has great potential for the treatment of SONFH.Objective1.To establish and evaluate a model of steroid-induced osteonecrosis of the femoral head;2.To study the antioxidant stress of porous nanocomposite Se@SiO₂ and its protective effect on oxidative stress in chondrocytes by in vitro culture of chondrocytes.3.To evaluate the efficacy of porous nanocomposite Se@SiO₂ in the treatment of steroid-induced osteonecrosis of the femoral head by animal experiments.Method1.Establishment and evaluation of SONFH model in rats:Twelve SPF and 12 weeks old male SD rats were randomly divided into two groups:experimental group?n=8?and control group?n=4?.After weighed,intraperitoneal injection of lipopolysaccharide 20ug/kg 2 times,each time interval of 24 hours,24 hours after intramuscular injection of methylprednisolone succinate?MPS?,20mg/kg,continuous injection 3 times,each interval 24 hours.The control group was given the same site to the same dose of saline.After 8 weeks of continuous feeding,the bilateral femoral head was taken and fixed in 4%paraformaldehyde solution for 24 hours.Microscopic CT scan and HE staining were used to evaluate the necrosis of femoral head.2.Chondrocyte culture and identification:The rats were sacrificed at the age of 3-4 weeks.The rats were sacrificed by anesthesia and 75%alcohol was immersed for 5 min.The bilateral knee and hip cartilage were isolated from the sterile condition.In the serum-free medium,Connective tissue membrane removed clean.The cartilage was cut into 1mm size by micrograph.Add 3%collagenase.Add the incubator at 37? for 45min,then add 5 times volume serum-free medium and incubate at 37? for 12h.?10%FBS,DMEM/F-12,1%antibiotics?after the supernatant was discarded and the filter was centrifuged.After centrifugation,the medium was centrifuged at 1000r/m for 3 min.,Planted in T75 culture bottle.The next day for liquid,about 6-7d for passage.The chondrocytes were used as 4-5 generation cells,and were identified by immunofluorescence staining with type II collagen.The cartilage was removed and EDTA was decalcified.The paraffin was embedded and stained with toluidine blue.3.Porous nanocomposites Se@SiO₂ inhibition of oxidative stress detection?ROS inhibition test?:Use the ROS test kit.The chondrocytes were seeded into 6-well plates?1×106 cells/well?.In the control group and the experimental group,the porous nanocomposites of the concentration of 40?M were added to the culture medium of the experimental group.Stimulate for 24 hours.The control group did not add.Then,1.5 ml of PBS containing 50 ?M H2O2 was added to each culture medium to stimulate the cells in the plate,placed in a 37?,5%CO2 incubator for 30 minutes,then washed twice with PBS and then added to the working concentration of DCFH-DA.After staining at 37? for 15 minutes,the plate cells were recorded with a fluorescence microscope.4.Protective effect of Porous nanocomposites Se@SiO₂ on oxidative stress in chondrocytes:Use the TUNEL kit?Roche,USA?,according to the kit instructions.The cultured chondrocytes were divided into H2O2 group?control group?,H2O2 + Se@SiO₂ group?experimental group?.In the experimental group,the Porous nanocomposites Se@SiO₂ was added at a concentration of 40 ?M and pre-stimulated for 24 hours.The cells were then incubated for 24 hours with 4%paraformaldehyde for 15 min,washed three times with PBS for 5 min,and Triton X-100 was allowed to pass through for 2 minutes.The samples were then incubated in a 50 ?L TUNEL reaction mixture at 37? for 1 hour,stained with DAPI and observed by fluorescence microscopy.The apoptotic rate was the ratio of the number of TUNEL positive cells to the total number of cells in the field of view,and 20 visual fields were statistically analyzed.5.Experiment on the treatment of SONFH with Porous nanocomposites Se@SiO₂.A total of 36 SPF,12 weeks old and male SD rats were randomly divided into 3 groups:control group?group A?,simple hormone-induced group?group B?,hormone-induced+ Porous nanocomposites Se@SiO₂ treatment group?group C?.All animals are adapted for one week.B group,group C the first day,intraperitoneal injection of lipopolysaccharide 20ug/kg,injection 2 times,each time interval of 24 hours,24 hours after intramuscular injection of methylprednisolone succinate?MPS,the United States,Pfizer?,40mg/Kg,continuous injection 3 times.Seven days after the last injection of MPS,Group C was given intraperitoneal injection of 1 mg/kg of Porous nanocomposites Se@SiO₂ at intervals of 24 hours for 14 days and group B was given equal doses of saline.Group A was given equal doses of saline at the same site and at the same time.At the 8th week after the first administration,all the SD rats were sacrificed by anesthesia,and the bilateral femoral head specimens were taken into the 4%paraformaldehyde solution for more than 24 hours.The samples were then subjected to Micro CT scan and HE staining.result:1.Experimental modeling of steroid-induced femoral head necrosis in SD rats:The CT analysis showed that the bone mineral density,trabecular bone thickness and trabecular bone separation were significantly lower in the model group than in the control group.HE staining was used to observe the trabeculae fracture,bone marrow necrosis,empty pit of bone formation and increased number of fat cells,increase,partly integrated into a bubble2.Chondrocyte culture identification results:Extraction of the cartilage used in the toluidine blue staining was positive,cell type II collagen was stained positive.3.Porous nanocomposites Se@SiO₂ inhibition of oxidative stress detection?ROS inhibition test?Results:ROS expression is indicated by the fluorescence intensity of DCFH?green?.After H2O2 intervention,the expression of pre-stimulated ROS in the chondrocytes of the experimental group was significantly lower than that of the control group.4.Protective effect of Porous nanocomposites Se@SiO₂ on oxidative stress in chondrocytes:TUNEL staining positive for green fluorescent highlights for damage to apoptotic cells.A large number of green fluorescent bright spots were detected in the H2O2 stimulation group,and the green fluorescent light was detected by the Porous nanocomposites Se@SiO₂ group.The statistical analysis was P<0.05,which was statistically significant.5.The results of Porous nanocomposites Se@SiO₂ treatment the SONFH in Rats:HE results showed that bone trabeculae fracture,bone marrow necrosis,cranial lacunae formation and Hypertrophy,an increase in fat cells were observed in group B?model group?.Group C?porous nanocomposite Se 1b group?was significantly less than that in group B?P<0.05?,and between group A?control group?and group B.Micro CT scan and three-dimensional statistical analysis showed that:severe cases of femoral head necrosis group B>C group>A group.conclusions:1.Lipopolysaccharide?LPS?combined with methylprednisolone succinate?MPS?to establish rat model of steroid-induced femoral head necrosis,the success rate is high,safe and feasible;2.The cultured cells were chondrocytes;3.Porous nanocomposites Se@SiO₂ can antioxidant stress;4.Porous nanocomposites Se@SiO₂ can protect the chondrocytes to avoid oxidative stress injury;5.Porous nanocomposites Se@SiO₂ through the role of antioxidant stress,can treat steroid-induced femoral head necrosis.