| Part Ⅰ:The effect of COX-2 inhibitor on subchondral bone in mice during the process of DMM model buildingObjective:To investigate the effect of COX-2 inhibitor on subchondral bone in mice during the process of DMM model building.Methods:Fifty 8-week-old male C57BL/6 mice were randomly divided into 5 groups with 10 mice in each group:Sham group,osteoarthritis model+vehicle injection group(DMM+veh group),osteoarthritis model+5mg/kg COX-2 inhibitor injection group(DMM+C5 group),osteoarthritis model+10mg/kg COX-2 inhibitor injection group(DMM+C10 group)and osteoarthritis model+20mg/kg COX-2 inhibitor injection group(DMM+C20 group).Medial meniscotibial ligament was cut to build the DMM model.After surgery,the mice were intraperitoneally injected with 0.1ml vehicle solution,5mg/kg COX-2 inhibitor,10mg/kg COX-2 inhibitor and 20mg/kg COX-2 inhibitor three times a week.The procedure of sham group was as same as DMM group without transection of medial meniscotibial ligament.Four weeks later,the mice were euthanized and knee joint specimens were collected for micro-CT,AFM analysis,HE staining and safranin O-fast green staining.Results:Micro-CT analysis showed that the bone mass in the DMM+veh group significantly increased compared to sham group,which was considered as "osteosclerosis".However,the bone mass in the subchondral bone decreased in DMM mice treated with COX-2 inhibitor compared with the DMM+veh group and sham group.AFM analysis showed that compared with sham group,the elastic modulus of subchondral bone in the DMM+veh group was reduced and the elastic modulus of subchondral bone was further reduced in the DMM+COX-2 inhibitor group.The results of histopathological staining showed that the cartilage surface of the sham group was complete,the cartilage morphology was good,the proteoglycan loss of articular cartilage and structural damage were not obvious,and the morphology and staining of cartilage cells were normal.In the DMM+veh group,some proteoglycans were lost in the superficial layer of articular cartilage,the cartilage surface was rough,discontinuous and irregular.There was no significant difference in cartilage and synovitis between DMM+veh group and DMM mice treated with COX-2 inhibitor.Conclusion:At some extent,Cox-2 inhibitor could affect the remodeling of subchondral bone and reduce the osteosclerosis of the subchondral bone in mice during the process of DMM model building.Part Ⅱ:The effect of tramadol on subchondral bone in mice during the process of DMM model buildingObjective:To investigate the effect of tramadol on subchondral bone in mice during the process of DMM model building.Methods:Fifty 8-week-old male C57BL/6 mice were randomly divided into 5 groups with 10 mice in each group:Sham group,osteoarthritis model+vehicle injection group(DMM+veh group),osteoarthritis model+10mg/kg tramadol injection group(DMM+T10 group),osteoarthritis model+20mg/kg tramadol injection group(DMM+T20 group)and osteoarthritis model+40mg/kg tramadol injection group(DMM+T40 group).Medial meniscotibial ligament was cut to build the DMM model.After surgery,the mice were intraperitoneally injected with 0.1ml vehicle solution,10mg/kg tramadol,20mg/kg tramadol and 40mg/kg tramadol three times a week.The procedure of sham group was as same as DMM groups without transection of medial meniscotibial ligament.Four weeks later,the mice were euthanized and knee joint specimens were collected for micro-CT,AFM analysis,HE staining and safranin O-fast green staining.Results:Micro-CT analysis showed that the bone mass in the DMM+veh group significantly increased compared to sham group,which was considered as "osteosclerosis".It also showed that the BV/TV in the DMM+veh group increased compared to sham group.However,the BV/TV in the subchondral bone decreased in DMM mice treated with tramadol compared with the DMM+veh group.AFM analysis showed that compared with sham group,the elastic modulus of subchondral bone in the DMM+veh group was reduced and the elastic modulus of subchondral bone was further reduced in the DMM+T group.The results of histopathological staining showed that the cartilage surface of the sham group was complete,the cartilage morphology was good,the proteoglycan loss of articular cartilage and structural damage was not obvious,and the morphology and staining of cartilage cells were normal.In the DMM+veh group,some proteoglycans were lost in the superficial layer of articular cartilage,the cartilage surface was rough,discontinuous and irregular.There was no significant difference in cartilage between DMM+T10 group,DMM+T20 group and DMM+T40 group.HE staining results of synovium showed that synovitis was worse in DMM+veh group than in sham group.Compared with DMM+veh group,synovitis scores in DMM+T10 and DMM+T20 groups showed no difference.Conclusion:Tramadol has little effect on the articular cartilage in DMM mice.At some extent,it could affect the remodeling of subchondral bone and reduce the osteosclerosis of the subchondral bone in mice during the process of DMM model building.Part Ⅲ:To investigate the effect of COX-2 inhibitor or tramadol combined with alendronate sodium on subchondral bone in mice during the process of DMM model buildingObjective:To investigate the effect of COX-2 inhibitor or tramadol combined with Alendronate sodium on subchondral bone in mice during the process of DMM model building.Methods:Sixty 8-week-old male C57BL/6 mice were randomly divided into 6 groups with 10 mice in each group:Sham group,osteoarthritis model+vehicle injection group(DMM+veh group),osteoarthritis model+COX-2 inhibitor injection+vehicle injection group(DMM+C20+veh group),osteoarthritis model+COX-2 inhibitor injection+alendronate sodium injection group(DMM+C20+A40 group),osteoarthritis model+tramadol injection+vehicle injection group(DMM+T10+veh group)and osteoarthritis model+tramadol injection+alendronate sodium injection group(DMM+T10+A40 group).Medial meniscotibial ligament was cut to build the DMM model.After surgery,the mice were intraperitoneally injected with 0.1ml vehicle solution,20mg/kg COX-2 inhibitor,10mg/kg tramadol and 40mg/kg alendronate sodium three times a week.The procedure of sham group was as same as DMM groups without transection of medial meniscotibial ligament.Four weeks later,the mice were euthanized and knee joint specimens were collected for micro-CT,AFM analysis,HE staining and safranin O-fast green staining.Results:Micro-CT analysis showed that the bone mass in the DMM+veh group increased compared to sham group.However,the bone mass in the subchondral bone decreased in the DMM+C20+veh group compared with the DMM+veh group.After alendronate sodium treatment,the BV/TV in the DMM+C20+A40 group increased(p<0.05).Similarly,the bone mass in the DMM+T10+A40 group increased compared with DMM+T10+veh group(p<0.05).AFM analysis showed that compared with sham group,the elastic modulus of subchondral bone in the DMM+veh group was reduced and the elastic modulus of subchondral bone was further reduced in the DMM+C20+veh group.After alendronate sodium injection,the elastic modulus of the subchondral bone in the DMM+C20+A40 group increased compared with the DMM+C20+veh group(p<0.05).Similarly,the elastic modulus of subchondral bone in the DMM+T10+A40 group increased compared with the DMM+T10+veh group(p<0.05).The results of histopathological staining showed that the cartilage surface of the sham group was complete,the cartilage morphology was good,the proteoglycan loss of articular cartilage and structural damage were not obvious,and the morphology and staining of cartilage cells were normal.In the DMM+veh group,some proteoglycans were lost in the superficial layer of articular cartilage,the cartilage surface was rough,discontinuous and irregular.There was no significant difference in cartilage between DMM+C20+veh group and DMM+C20+A40 group as well as DMM+T10+veh group and DMM+T10+A40 group.Synovitis scores in DMM+C20+A40 group and DMM+T10+A40 group were slightly lower than those in DMM+C20+veh group and DMM+T10+veh group.Conclusion:Alendronate sodium combined with COX-2 inhibitor or tramadol could increase subchondral bone mass and improve biomechanical properties of the subchondral bone during the process of DMM model building.In addition,Alendronate sodium combined with COX-2 inhibitor or tramadol could partially reduce synovitis in DMM mice,and has no significant effect on articular cartilage. |
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