Effect Of Adrenergic Signal On Osteoblasts Induced By Ti Particles And Its Effect On Bone Resorption

Objective:To study the mechanism of adrenergic receptor signaling in osteolysis around the prosthesis through animal and eytological experiments and to preliminarily explore the role of exo somes in osteolysis induced by wear particles.Methods:In animal experiments:A mouse skull model was firstly established with Ti particles:which was divided into(A)CONTROL group(Sham group);(B)Ti group(Surgery group);(C)Ti+CLEN group(Surgery and intraperitoneal injection of lmg/kg clenbuterol);(D)Ti+PROP-LOW(Surgery and intraperitoneal injection of 0.1mg/kg propranolol);(E)Ti+PROP-HIGH(Surgery and intraperitoneal injection of 5mg/kg propranolol)。After weight changes of mice were measured:skull models were collected after death for micro-CT scanning,H&E staining and TRAP staining.2.In vitro experiments were conducted on MC 3T3-E1 cells,to detect the effects of β2 adrenergic receptors and osteoblast-osteoclast related molecules induced by Ti particles in osteoblasts by RT-PCR,Western blot and immunofluorescence staining.3.To collect exosomes of osteoblasts were stimulated by wear particles.then identified exosomes by transmission electron microscopy,NTA and Western blot.The expression of RANKL protein of exosomes and cells was determined by Western blot.Results:1.No abnormal death occurred in mice during the in experiment.3D image reconstruction and micro-CT results indicated that Ti group decreased BMD,BV/TV Tb.Th and increased Tb.Sp(P<0.05).Clenbuterol further reduced BMD BV/TV,Tb.Th:and increased Tb.Sp(P<0.05).While propranolol increased BMD BV/TV.Tb.Th and decreased Tb.Sp(P<0.05).H&E and TRAP staining results showed that Ti group induced osteoblastic depressions and osteoclast cell number.while Ti+CLEN group further aggravated the depressions and showed more osteoclast positive cells.Both Ti+PROP groups significantly reduced the area of bone erosion and the number of osteoclasts on the skull surface.2.In vitro experiments showed that Ti particles increased the expression ofβ2-AR on the surface of MC3T3-E1 cells,which peaked at 0.01mg/mL.PCR results showed that both Ti particles and Clenbuterol increased RANKL mRNA expression(P<0.05)and they had a synergistic effect,further increasing RANKL expression(P<0.01).In Ti+CLEN+PROP group,RANKL mRNA significantly decreased while OPG mRNA increased(P<0.05).In Ti group,COX-2 mRNA was significantly increased compared with CONTROL group,but adrenal drugs had no significant effect on COX-2 mRNA(P>0.05).Then,immunofluorescence staining experiments were conducted to verify that the expression of RANKL was significantly increased in Ti group and CLEN group compared with CONTROL group,while propranolol sigificantly decreased the expression of RANKL 3.Track-like morphology of extracellular vesicles extracted was observed by transmission electron microscopy.NTA showed that the diameter was between 40-150nm.Western blot showed high expression of TSG101,CD81 and low expression of GAPDH.Moreover,the exosomes labeled with PKH67-green fluorescence were observed in BMM cells.Then,Western blot results showed that RANKL was highly expressed in osteoblasts and exosomes induced by 0.01mg/ml titanium particles.Finally,exosomes were added to BMM cells which treated by M-CSF and RANKL.TRAP staining showed no significant difference in the mumaber of osteoclasts in the CONTROL-EXO group(215.33±8.73/well)and CONTROL group(207±12.28/well).The number of osteoclasts in the Yi-0.01mg/ml-EXO group(302.66±40.8/well)increased significantly compared with the other two groups(P<0.05).Conclusion:1.In the mouse model,propranolol can dose-dependently inhibit the bone surface depression caused by wear particles and reduce the number of osteoclasts,it has a significant anti-bone absorption effect.2.Titanium granule stimulates the increased expression of the surface of osteoblast cells and promotes the activation of the β2-adrenergic signaling pathway.thereby significantly increasing the generation of RANKL,resulting in rapid bone mass loss.Otherwise propranolol can sigificantly inhibit the expression of RANKL in osteoblasts induced by Ti particles to inhibit osteoclast activity and then reduce bone resorption.3.Ithas been preliminarily proved that titanium particles which can promote bone resorption by stimulating osteoblasts to release exosomes with high expression of RANKL.