Trichostatin A Enhancing Osseointegration Of Titanium Rods In Osteoporotic Rats

Objective: To study the ability of trichostatin A(TSA)to activate AKT/Nrf2 pathway signaling and to thereby improve the osseointegration of titanium rods by suppressing oxidative stress.Methods: Both in vivo and in vitro experiments were used in this study.In vivo experiment: ovariectomized rat titanium screw implantation model.Sixty 12-week-old female rats were divided into Sham group(n=15,Sham operation)and OVX group(n=45,bilateral oophorectomy).After 12 weeks,5 rats were selected from each group to determine whether the animal model of osteoporosis was successfully established.After confirming the establishment,bone defects were operated on the remaining rats,and titanium rods were implanted in 20 randomly selected rats from the remaining 40 rats in the OVX group.The rats were divided into 5 groups: CON group(n=10,sham operation,bone defect operation);BD group(n=10,OVX,bone defect surgery);TSA group(n=10,OVX,bone defect surgery,TSA intraperitoneal injection);TI group(n=10,OVX,bone defect surgery,titanium rod implantation);TSA+TI group(n=10,OVX,bone defect surgery,titanium rod implantation,intraperitoneal injection of TSA).All rats were sacrificed 8 weeks later.The femur samples of rats were examined by micro-CT,H&E staining,Masson trichroic staining and nail pulling test.BMSCs were taken for mineralization induction and alkaline phosphatase activity and osteogenic ability were measured.In vitro experiment: CCCP induced MC3T3-E1 cell oxidative stress model was used.Firstly,TSA antagonized osteoblast injury induced by oxidative stress.The cells were divided into 6 groups: NC group;CCCP group;TSA Group;CCCP+TSA group;TSA+LY294002 group;CCCP+TSA+LY294002 group.MC3T3-E1 cells after different treatments were tested by MMP,oxidative stress index,apoptosis,and Western blot.Then,nuclear protein and cytoplasmic protein were respectively extracted from CCCP group,CCCP+TSA group and CCCP+TSA+LY294002 group,and the expression levels of Nrf2 protein in and outside the nucleus were detected by Western blot.Next,MC3T3-E1 cells were added nano titanium powder,and the cells were divided into four groups: CCCP group;CCCP+TI group;CCCP+TI+TSA group;CCCP+TI+TSA+LY294002 group.ALP staining was performed on MC3T3-E1 cells after different treatments to observe the fusion of cells with nano-sized titanium powder and alkaline phosphatase activity.Finally,MC3T3-E1 cells were planted on germ-free titanium sheet and divided into three groups: TS+CCCP group;TS+CCCP+TSA group;TS+CCCP+TSA+LY294002 group.MC3T3-E1 cells after different treatments were scanned by electron microscope,and the adhesion of cells to titanium plate was observed.Results: TSA ultimately improved distal femur trabecular bone microstructural characteristics,increased BMSC mineralization capacity,improved alkaline phosphatase activity,promoted bone formation,and contributed to the improved osseointegration of titanium rods in vivo.In vitro,TSA treatment of CCCP-treated MC3T3-E1 cells resulted in the upregulation of osteogenic proteins including OPN,Runx2,BMP2,and OCN in MC3T3-E1 together with increased p-AKT,total Nrf2,nuclear Nrf2,HO-1,and NQO1 expression,enhanced mitochondrial functionality,and decreased oxidative damage.Notably,the PI3K/AKT inhibitor LY294002 reversed these effects.Conclusion: TSA was able to reverse oxidative stress-induced cell damage while promoting the healing of bone and improving the osseointegration of titanium rods through AKT/Nrf2 pathway activation.