The Mechanism Study For The Treatment Of Osteonecrosis Of The Femoral Head Through Regulating The Differentiation Of Stem Cells And Endoplasmic Reticulum Stress

BackgroundOsteonecrosis of the femoral head(ONFH)is a serious orthopedic problem,which primarily results from ischemia/hypoxia to the femoral head.In the process of normal bone metabolism,bone marrow stem cells can develop into different directions.Osteoblasts from stem cells is contribute to bone formation,and osteoclasts is beneficial to bone resorption.Bone formation and bone resorption complement each other to form the normal bone metabolism process.In ONFH,due to ischemia/hypoxia,bone metabolism process and the differentiation of bone marrow stem cells are likely to be disturbed.In addition,one of cellular manifestation induced by ischemia/hypoxia is endoplasmic reticulum(ER)stress.However,the relationship among ONFH,ER stress and the differentiation of bone marrow stem cells were not clear.Joint loading is one form of non-invasive physical treatment.Joint loading has been applied to synovial joints such as the elbow,knee and ankle.Our bone histomorphometric studies demonstrated that knee loading stimulated bone formation.We have also demonstrated that knee loading could accelerate the healing of surgical wounds in the femoral neck and tibia.In addition,salubrinal is a synthetic chemical that inhibits de-phosphorylation of eIF2a,and it reduces cell death from the ER stress at a proper dose.In our previous studies,salubrinal stimulates bone healing,and serves as a potential drug candidate for treatment of osteoarthritis and osteoporosis by reducing joint inflammation and bone loss.Knee loading and salubrinal improve bone metabolism in several animal models,but its effects on osteonecrosis have not been investigated.ObjectivesTo understand possible linkage of ONFH to ER stress and the differentiation of bone marrow stem cells,and to find more effective therapies,a surgery-induced animal model was employed.We examined a hypothesis that mechanical loading and salubrinal enhanced vessel remodeling and bone healing through the modulation of the fate of bone marrow-derived cells,and salubrinal was used as an agent to evaluate the role of ER stress in ONFH.MedthosIn the first study,eighteen rats were randomly divided into 3 groups: sham operated control group(Sham,n = 6),osteonecrosis group(ON,n = 6),and mechanical loading treated osteonecrosis group(ON+loading,n = 6).ONFH was induced by transecting the ligamentum teres followed by a tight ligature around the femoral neck.For knee loading,5 N loads were laterally applied to the knee at 15 Hz for 5 min/day for 5 weeks.Changes in bone mineral density(BMD)and bone mineral content(BMC)of the femur were measured by pDEXA,and ink infusion was performed to evaluate vessel remodeling.Femoral heads were harvested for histomorphometry,and bone marrow-derived cells were isolated to examine osteoclast development and osteoblast differentiation.In the second study,sixty rats were randomly divided into 4 groups: sham control group(Sham,n = 18),osteonecrosis group(ON,n = 18),salubrinal-treated osteonecrosis group(ON+S,n = 18)and salubrinal-treated sham control group(Sham+S,n = 6).Ischemic osteonecrosis of the bilateral femoral heads were induced according to the procedure previously described.Trabecular structural parameters(BV/TV,Tb.N,Tb.Th and Tb.Sp),BMD and BMC of the femur head were measured by μ-CT and pDEXA.Ink infusion was performed to evaluate vessel remodeling.Femoral heads were harvested for histomorphometry,and bone marrow-derived cells were isolated to examine osteoclast development and osteoblast differentiation.The expression levels of p-eIF2α,ATF4,GRP78,CHOP,VEGF and NFATc1 of femoral heads samples in vivo were determined by immunofluorescence and western blot assays.The viability,migration,tube formation and western blot assays of endothelial cells in vitro were performed to investigate whether salubrinal affected angiogenesis against ER stress.In addition,cell viability,osteoblast(MC3T3-E1)markers(ALP and RUNX2)and the ER stress signaling(p-e IF2α,ATF4)were examined in vitro to evaluate whether salubrinal improved osteogenesis against ER stress.ResultsThe results showed that ONFH significant induced bone loss.In the ON group,the values of BV/TV,Tb.N,Tb.Th,B.Ar/T.Ar and BMD/BMC were decreased,and the value of Tb.Sp was increased.However,knee loading and salubrinal treatment significantly increased the values of BV/TV,Tb.N,Tb.Th and B.Ar/T.Ar,as well as the change of femoral BMD/BMC.It also decreased the value of Tb.Sp.ONFH induced the reduction in vessel perfusion,and knee loading and salubrinal treatment improve them.For bone marrow-derived cells assays,ONFH activated osteoclast development.Mechanical loading and salubrinal treatment reduced its formation,migration,adhesion and the level of “pit” formation.Furthermore,knee loading and salubrinal significantly increased osteoblast differentiation and CFU-F.In addition,the salubrinal-treated group also increased the level of p-e IF2α,ATF4,GRP78,and VEGF,but it presented a lower level of NFATc1 than that the ON group in vivo.Moreover,salubrinal also protected osteoblast development under ER stress condition by upregulating the levels of ATF4,ALP and RUNX2 in vitro.Furthermore,it stimulated angiogenesis of endothelial cells against ER stress through elevating the levels of p-e IF2α in vitro.ConclusionsThe current study suggests that ONFH induces osteonecrosis,bone loss,reduction in vessel perfusion and excessive osteoclastogenesis in the femoral head.Mechanical loading and salubrinal treatment are effective in repair osteonecrosis of the femoral head in a rat model.The effects of mechanical loading and salubrinal might be attributed to promoting vessel remodeling,suppressing osteoclast development,and increasing osteoblast and fibroblast differentiation by modulating the fate of bone marrow-derived cells.In addition,the results support the notion that ER stress may be an important pathological mechanism in the surgery-induced ONFH model,and salubrinal improves ONFH symptoms by enhancing angiogenesis and bone healing via suppressing the ER stress.In summary,mechanical loading and salubrinal might potentially be employed as new therapies for osteonecrosis of the femoral head.