Experimental Studies On The Three-dimensional Structure And Mechanical Property Of Subchondral Bone In Osteoarthritis And The Intervention Effect Of Diphosphonate

PartⅠ Three-dimensional structure variationof subchondral bone andthe intervention effect of diphosphonate in osteoarthritis.Objective Observing the Three-dimensional structure changes of subchondral bone ofthe rabbit instable knee joint at its early stage and the intervention effect ofdiphosphonate， to investigate the role of Three-dimensional structure changes ofsubchondral bone at the early stage of osteoarthritis in initiation and progression ofosteoarthritis.Methods60male New Zealand white rabbits were assigned randomly into threegroups according to random digits table: the control group (n＝12), the model group (n＝24). the diphosphonate group (n＝24). OA model of rabbit instable knee joint (anteriorcruciate ligament transection，ACLT) was achieved in the right knee joint.The rabbits inthe diphosphonate group were injected by sub-cutaneous (s.c.) way with risedronate at thedosage of0.01mg per kg body mass daily immediately after surgery;The rabbits in thecontrol group and in the the model group were injected by sub-cutaneous (s.c.) way withnormal saline of the same volume at the same time.8rabbits from the model group,8rabbits from the diphosphonate group and4rabbits from the control group were sacrificedby aeroembolism at4week、8week and12week following surgery. The right knee wasdissected and scanned by micro-computed tomography to assess morphology and density.Results Compared with control group, the bone volume f raction(BV/TV)，thetrabecula r number (Tb. N) and trabecular thickness (Tb.Th) in model group and diphosphonate group were reduced remarkably at4week after operation (P<0.01)．Thebone volume f raction(BV/TV) was reduced remarkably in model group than that indiphosphonate group(P<0.05). BVF in diphosphonate group were obviously lower thanthat in the control group(P<0.05). Compared with control group, the trabecularseparation (Tb. Sp) in model group and diphosphonate group was obviouslylarger(P<0.01). Bone mineral density(BMD) in model group was obviously lower than thatin the control group and diphosphonate group (P<0.01) and no significant difference wasnoted between the control group and diphosphonate group(P>0.05). At12week afteroperation, Compared with control group and diphosphonate group, the bone volume fraction(BV/TV)，the trabecula r number (Tb. N) and trabecular thickness (Tb.Th) inmodel group was were significantly higher(P<0.05)．The trabecular separation (Tb. Sp)was reduced remar kably in model group than that in the control group and diphosphonategroup(P<0.01). We also compared bone microstructure index between12week afteroperation and those at4week after operation. The bone volume f raction(BV/TV)，thetrabecula r number (Tb. N) and trabecular thickness (Tb.Th) in model group was weresignificantly higher(P<0.01). The trabecular separation (Tb. Sp) in model group anddiphosphonate group was obviously larger(P<0.01). Bone mineral density(BMD) in modelgroup at12week after operation was obviously increased at4week after operation(P<0.01).Conclusions In the early stage of the instable knee，three-dimensional structure ofsubchondral bone were destroyed remarkably. Bone microstructure index weresignificantly lower and showing obviously bone remodeling. The damages of bonemicrostructure were seen firstly and showed remarkably bone formation Later. Bonemicrostructure index were markedly higher. Diphosphonate may improvethree-dimensional structure and mechanical properties of subchondral bone greatly，whichis associated with inhibiting subchondral bone resorption.Three-dimensional structurechanges of subchondral bone play a important role in the initial development of OA. PartⅡ Finite Element Analysis of Biomechanical variation ofsubchondral bone and the intervention effect of diphosphonate inosteoarthritis.Objective： Observing the biomechanical changes of subchondral bone of the rabbitinstable knee joint at its early stage and the intervention effect of diphosphonate，toinvestigate the role of biomechanical changes of subchondral bone at the early stage ofosteoarthritis in initiation and progression of osteoarthritis.Methods60male New Zealand white rabbits were assigned randomly into three groupsaccording to random digits table: the control group (n＝12), the model group (n＝24). thediphosphonate group (n＝24). OA model of rabbit instable knee joint (anterior cruciateligament transection，ACLT) was achieved in the right knee joint.The rabbits in thediphosphonate group were injected by sub-cutaneous (s.c.) way with risedronate at thedosage of0.01mg per kg body mass daily immediately after surgery;The rabbits in thecontrol group and in the the model group were injected by sub-cutaneous (s.c.) way withnormal saline of the same volume at the same time.8rabbits from the model group,8rabbits from the diphosphonate group and4rabbits from the control group were sacrificedby aeroembolism at4week、8week and12week following surgery. The right knee wasdissected and scanned by micro-computed tomography to assess morphology anddensityand then converted to micro-finite element models．Using Mimics software andANSYS finite element software，we establish the knee joint three-dimensional finiteelement model. Specimen-specific finite element analyses quantified the periarticular bonearchitecture and mechanical properties.Results Compared with control group and diphosphonate group, the bone volume fraction(BV/TV)，elastic module(EM), reaction force to1%strain (RF), mean von Misesstress (VM), in model group were reduced remarkably at4week after operation(P<0.01)．These was lower in diphosphonate group than that in the control group and no significant difference was noted between the control group and diphosphonategroup(P>0.05). Bone mineral density(BMD) in model group was obviously lower than thatin the control group and diphosphonate group (P<0.01) and no significant difference wasnoted between the control group and diphosphonate group(P>0.05). At12week afteroperation, Compared with control group and diphosphonate group, the bone volume fraction(BV/TV)，bone mineral density(BMD) in model group were increased remarkablyat4week after operation (P<0.01)．Elastic module(EM), reaction force to1%strain (RF),mean von Mises stress (VM) in model group were obviously lower than that in the controlgroup and diphosphonate group (P<0.01) and no significant difference was noted betweenthe control group and diphosphonate group(P>0.05). We also compared bonemicrostructure index and mechanical properties between12week after operation and thoseat4week after operation. The results showed the bone volume f raction(BV/TV)，elasticmodule(EM), reaction force to1%strain (RF), mean von Mises stress (VM) at12week inmodel group after operation were obviously lower than that at4week after operation(P<0.01)．And we did correlation comparison, results showed that4weeks after operation,the correlation coefficient between elastic modulus and the Mankin scorewas―0.835(r=―0.835),(P<0.01). There was a significant negative correlation. Thecorrelation coefficient between elastic modulus and bone density was0.848(r=0.848),(P<0.01), there was significant positive correlation. The correlation coefficient betweenelastic modulus and volume fraction was0.893(r=0.893),(P<0.01), there was significantpositive correlation.At12weeks, The correlation coefficient between elastic modulus andthe Mankin score was―0.883(r=―0.883),(P<0.01), there was significant negativecorrelation. The correlation coefficient between elastic modulus and bone density was0.861(r=0.861),(P<0.01), there was significant positive correlation; The correlationcoefficient between elastic moduli and the bone volume f raction(BV/TV) was0.817(r=0.817)，（P<0.01）, there was significant positive correlation.Conclusions In the early stage of the instable knee，biomechanical changes ofsubchondral bone were occured. Elastic module of subchondral bone greatly decreased, which were connected with subchondral bone resorption resulting from microfracture byabnormal stress in the early phase. Diphosphonate may improve elastic module ofsubchondral bone greatly，which is associated with inhibiting subchondral bone resorption.Biomechanical changes of subchondral bone play a important role in the initialdevelopment of OA.. PartШ Angiogenesis at the osteochondral junction and the interventioneffect of diphosphonate in instable knee joint of early stageObjective：We investigated angiogenesis at the osteochondral junction of knees withosteoarthritis,and the inhibition effects of diphosphonate at the early stage of instable kneejoint. This study aimed to characterise animal models of knee OA with particular respect toosteochondral angiogenesis and display different relationships with disease severity andreveal the mechanism.Methods60male New Zealand white rabbits were assigned randomly into three groupsaccording to random digits table: the control group (n＝12), the model group (n＝24). thediphosphonate group (n＝24). OA model of rabbit instable knee joint (anterior cruciateligament transection，ACLT) was achieved in the right knee joint.The rabbits in thediphosphonate group were injected by sub-cutaneous (s.c.) way with risedronate at thedosage of0.01mg per kg body mass daily immediately after surgery;The rabbits in thecontrol group and in the the model group were injected by sub-cutaneous (s.c.) way withnormal saline of the same volume at the same time.8rabbits from the model group,8rabbits from the diphosphonate group and4rabbits from the control group were sacrificedby aeroembolism at4week、8week and12week following surgery. Then the femurcondyles of right knee were harvested.Specimens were processed for gross morphologicexamination, mankin score analysis detection and immunohistochemical analysis of articular cartilage. Vessels at the osteochondral junction were identified byimmunohistochemistry and quantified by computer-assisted image analysis. Diseaseseverity was assessed using a scoring system.Results There was no detectable macroscopic change in the appearance of the joint inany groups at week after surgery.At12week,the diphosphonate group exhibited a roughsurface and no osteophyte formation in the medial femural condyles.There was a roughsurface and significant osteophyte formation in the margin of the medial femoral condylesin model group. Compared with control group,Mankin score of model group increasedmarkedly at4week and12week after operation (P<0.01).But only at12week,the mankinscore in diphosphonate group increased significantly(P<0.05). Diphosphonate can greatlyinhibit the injury of articular cartilage. Compared with model group, mankin score in thediphosphonate group decreased significantly(P<0.05). There was a significant increase ofangiogenesis in model group at4week and12week postoperation (P<0.05) compared withcontrol group..But diphosphonate group decrease significantly in angiogenesis at4weekand12week after surgery compared with those of model group(P<0.05). Compared withcontrol group, there was a significant increase in the number of angiogenesis in the modelgroup.at4week and12week after operation (P<0.01). Diphosphonate can inhibitangiogenesis at the osteochondral junction of knees with osteoarthritis. Compared withmodel group, diphosphonate group decreased markedly in the number of angiogenesis at4week and12week after operation(P<0.01). Osteochondral vascular density increased withincreasing cartilage severity and clinical disease activity scores, Osteochondral vascularityis associated with the severity of OA cartilage changes and clinical disease activity.Significant differences in vascularity were not observed between medial and lateralcompartments of rabbit knees.Conclusions Osteochondral vascularity is associated with the severity of OA.Oteochondral angiogenesis was demonstrated as increased vascular density innon-calcified articular cartilage in OA. Diphosphonate can inhibit angiogenesis at theosteochondral junction of knees with osteoarthritis. Modulation of osteochondral angiogenesis may differentially affect OA disease. Part IV Biochemistry change of in subchondral bone and theintervention effect of diphosphonate in instable knee joint of early stageObjective： Observing expression changes of matrix metalloproteinase-9(MMP-9)、cathepsin K(CK) of subchondral bone and matrix metalloproteinase-13(MMP-13) inarticular cartilage and the inhibition effects of diphosphonate at the early stage of instableknee joint.To prove subchondral bone resorption in the early phase of instable kneejoint,and investigate the mechanism.Methods60male New Zealand white rabbits were assigned randomly into threegroups according to random digits table: the control group (n＝12), the model group (n＝24). the diphosphonate group (n＝24). OA model of rabbit instable knee joint (anteriorcruciate ligament transection，ACLT) was achieved in the right knee joint.The rabbits inthe diphosphonate group were injected by sub-cutaneous (s.c.) way with risedronate at thedosage of0.01mg per kg body mass daily immediately after surgery;The rabbits in thecontrol group and in the the model group were injected by sub-cutaneous (s.c.) way withnormal saline of the same volume at the same time.8rabbits from the model group,8rabbits from the diphosphonate group and4rabbits from the control group were sacrificedby aeroembolism at4week、8week and12week following surgery. Then the medialfemoral condyles of right knee were harvested. Specimens were processed forimmunohistochemical analysis of MMP-9、CK and MMP-13.Results There were some positive cells of MMP-9and CK in the subchondral bone,MMP-13in articular cartilage in all groups at4week and12week after operation.But thenumber of positive cells in control group and diphosphonate group were fewer than that inthe model group.Compared with control group, there was a significant increase in thenumber and the percentage of positive cells of MMP-9and CK in model group at4week and12week after operation (P<0.01). Diphosphonate can inhibit the positive cellsexpression of MMP-9、CK and MMP-13. Compared with model group, diphosphonategroup decreased markedly in the number and the percentage of positive cells of MMP-9、CK and MMP-13at4week and12week after operation(P<0.01).Also subchondral boneresorption decreased gradually with time at the early phase of instable knee joint.Thenumber and the percentage of positive cells of MMP-9、CK MMP-13at12week decreasedsignificantly compared with those at4week (P<0.05).Conclusions Subchondral bone resorption at the early phase of instable knee joint isassociated with a increase in the synthesis of MMP-9and CK.The inhibition ofsubchondral bone resorption by diphosphonate relate to reduction in the synthesis ofMMP-9and CK derived from the osteoclasts at the same phase.