Evaluation Of Integrin αvβ3 Expression In Steroid-induced Osteonecrosis Of The Femoral Head In Rats By USMI

ObjectiveNeovascularization occurs with osteonecrosis of the femoral head,and ordinary ultrasound contrast agents can not specifically develop neovascularization.The purpose of this study was to evaluate the spatio-temporal changes of integrin αvβ3 during the occurrence and development of steroid-induced osteonecrosis of the femoral head(Steroid-induced osteonecrosis of the femoral head,SIONFH)by using self-assembled i RGD targeted ultrasound contrast agent and ultrasonic molecular imaging(Ultrasound molecular imaging,USMI),and to read out the quantitative parameters by time-intensity curve.To explore the diagnostic value of USMI in rat SIONFH by comparing with tissue sections.Methods1.i RGD peptide(DSPE-PEG2000-i RGD)was synthesized from i RGD peptide and distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000)by "mercapto-Maleimide" covalent linking method,and distearoylphosphatidylcholine(DSPC),DSPE-PEG2000 and DSPE-PEG2000-i RGD were synthesized by film-hydration method to synthesize i RGD targeted ultrasound contrast agent,which was filled with perfluoropropane inert gas.The particle size and imaging of i RGD targeted ultrasound contrast agent were characterized.The specific binding test of i RGD targeted ultrasound contrast agent to mouse brain microvascular endothelial cells(b End.3)was carried out in vitro,which was divided into Targeted group(i RGD targeted ultrasound contrast agent + b End.3 cells),Non-Targeted group(nontargeted contrast agent + b End.3 cells)and inhibition group(free i RGD lipopeptide + b End.3 cells + i RGD targeted contrast agents).The adhesion efficiency of contrast agents to integrin αvβ3 on the surface of b End.3 cells was detected in the three groups.2.12 healthy SD rats were selected and the model of steroid-induced osteonecrosis of the femoral head was established by lipopolysaccharide(LPS)combined with methylprednisolone(MPS),Intramuscular injection of penicillin sodium to prevent infection.3.Magnetic resonance imaging(Magnetic Resonance Imaging,MRI)technique was used to scan the femoral head of the model rats at the 1st,3rd and 5th week to detect the degree of osteonecrosis of the femoral head.4.i RGD targeted ultrasound contrast agent and non-target ultrasound contrast agent were randomly injected into rats through tail vein,and the expression of integrin αvβ3 was detected noninvasively by ultrasound molecular imaging.Immediately after imaging,the femoral head of the rat model of steroid-induced osteonecrosis was taken for tissue section,and the expression of integrin αvβ3 was verified by immune double staining.Results1.i RGD targeted ultrasound contrast agent was successfully prepared.The particle size of i RGD targeted ultrasound contrast agent is 1.21 ±0.83 μm.Under light microscope,the particle size distribution is uniform and there is no adhesion.The imaging effect in vitro is good.2.In vitro i RGD targeted ultrasound contrast agent adhesion to b End.3 cells,there was significant difference between the targeted group and the Non-targeted group(32.75 ±0.95 VS 0.17 ±0.10,P < 0.05),and between the targeted group and the inhibition group(32.75±0.95 VS 0.15 ±0.10,P < 0.05).3.In the first week,MRI showed no abnormal signs of osteonecrosis of the femoral head.In the third week,MRI showed abnormal signs of osteonecrosis of the femoral head in steroid-induced osteonecrosis of the model rats.In the fifth week,the degree of osteonecrosis of the femoral head in the model rats was aggravated.4.The dynamic evaluation results of ultrasonic molecular imaging showed that there was no significant difference in the targeted ultrasonic molecular signal intensity between the targeted group and the Non-targeted group in the first week(1.13±0.13 d B VS 0.73±0.31 d B,P > 0.05),but in the third week,the signal intensity of the targeted group was significantly higher than that in the Non-targeted group(5.60±1.31 d B VS 0.94 ±0.81 d B,P < 0.01).At the5 th week,the targeted ultrasonic molecular signal intensity difference in the targeted group was significantly higher than that in the Non-targeted group,and the difference was statistically significant(10.90±1.11 d B VS 2.98±1.58 d B,P < 0.01).In the targeted group,the difference targeted ultrasonic molecular signal intensity in the third week was significantly higher than that in the first week(P < 0.05),and reached the maximum in the fifth week compared with that in the first week and the third week,The difference was statistically significant(P < 0.05).Immunofluorescence double staining analysis further confirmed that the expression of integrin αvβ3 in neovascularization endothelial cells of steroid-induced osteonecrosis of the femoral head gradually increased with the progression of osteonecrosis of the femoral head.ConclusionSelf-assembled i RGD targeted ultrasound contrast agent can evaluate the expression of integrin αvβ3 in neovascularization endothelial cells during the occurrence and development of steroid-induced osteonecrosis of the femoral head in rats.Ultrasound molecular imaging is expected to become a new method for early evaluation of vascular lesions in osteonecrosis of the femoral head,which can be used to evaluate the course and prognosis of steroidinduced osteonecrosis of the femoral head.