骨性关节炎软骨下骨三维结构和力学性能改变及早期干预的实验研究
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摘要
第一部分骨关节炎早期软骨下骨三维结构变化及二磷酸盐的干预效应
目的观察兔膝关节不稳早期软骨下骨三维结构变化及二磷酸盐的干预作用，以探讨骨性关节炎(OA)早期软骨下骨三维结构的变化在OA发生发展中的作用。
方法健康雄性新西兰大白兔60只，随机数字表法分为模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。。采用兔关节不稳模型（切断前交叉韧带）右膝造模。分三组：二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重；模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。分别切取保留关节面上下各2cm骨的手术侧膝关节，清除软组织，后行Micro-CT检查。得具体测量参数：骨体积分数(BVF),、骨小梁厚度Tb.Th)、骨小梁间隔(Tb.Sp)、骨小梁数量(Tb.N)、体积骨密度(vBMD)、组织骨密度（tBMD），并进行统计学分析。
结果模型组、二磷酸盐组、对照组不同时期的骨密度及软骨下骨微结构参数变化。显示术后第4W三组比较，模型组体积分数（BVF）、骨小梁数量(Tb.N)、骨小梁厚度（Tb.Th）比对照组明显降低，P<0.01；体积分数(BVF)比二磷酸盐组也降低P<0.05；BVF二磷酸盐组与对照组比亦降低,P<0.05；骨小梁分离度（Tb.Sp）模型组比二磷酸盐及对照组均增大,P<0.01。二磷酸盐组比对照组明显增大,P<0.01。骨密度模型组比二磷酸盐及对照组明显降低,P<0.05。而二磷酸盐组与对照组无统计学差异,P>0.05。12周时三组比较，模型组体积分数（BVF）、骨小梁厚度（Tb.Th）、骨小梁数目(Tb.N)比二磷酸盐及对照组明显增加,P<0.05。骨小梁分离度明显减少,P<0.05。而骨密度显著增加,P<0.01。我们对不同时期相关微结构参数也进行了比较,统计学分析。4周时和12周时软骨下骨微结构及骨密度变化情况比较，骨体积分数，骨小梁厚度，骨小梁数目明显增加,P<0.01。骨小梁分离度明显减小,P<0.01。12周时的骨密度较4周时显著增加,P<0.01。
结论膝关节不稳早期软骨下骨三维结构发生明显改变，表现明显的骨重塑。早期以骨破坏为主，各种骨量及骨结构指标均下降。后期出现明显的骨形成，各种骨量及骨结构指标均增加。二磷酸盐可通过抗骨吸收明显抑制软骨下骨的骨重塑，缓解骨质破坏和吸收，保护软骨下骨的骨结构，进而保护软骨下骨的力学性能，从而保护关节软骨。软骨下骨三维结构的改变在OA发生发展中具有重要作用。
第二部分骨关节炎软骨下骨力学性能变化有限元分析及二磷酸盐的干预效应
目的观察兔膝关节不稳早期软骨下骨力学性能的改变及二磷酸盐的干预作用，以探讨骨性关节炎(OA)早期软骨下骨力学性能的改变在OA发生发展中的作用。
方法健康雄性新西兰大白兔60只，随机数字表法60只新西兰大白兔随机分成三组，模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。采用兔关节不稳模型（切断前交叉韧带）右膝造模。二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重，模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。分别切取保留关节面上下各2cm骨的手术侧膝关节，清除软组织，进行大体评分。后行Micro-CT检查。从Micro-CT得到DICOM式的二维图像文件。将获得的DICOM导入Mimics V10.01软件中进行拟合，并导入到Ansys V10中进行有限元处理。得到关节软骨组织损伤Mankin评分；通过Micro-CT对60个标本进行检查，得到体积分数（BVF）、骨小梁厚度（Tb.Th）骨小梁数目(Tb.N)，骨小梁分离度(Tb.Sp)，骨密度（BMD）有限元软件计算得到弹性模量(EM)，反应力(RF)，平均Von Mises应力。后进行统计学分析比较。
结果兔膝关节不稳造模术后4W时，模型组、二磷酸盐组、对照组三组比较体积分数、弹性模量、反应力、平均Von Mises应力参数均下降，模型组比二磷酸盐组、对照组低, P<0.01。二磷酸盐组与对照组也下降，但P>0.05。骨密度模型组比二
磷酸盐及对照组明显降低，而二磷酸盐组与对照组无统计学差异（P>0.05）。12W时
模型组体积分数（BVF）、骨密度（BMD）明显增加, P<0.01。弹性模量、反应力，
Von Mises应力较对照组和二磷酸盐组低;二磷酸盐组亦较对照组低，但无统计意义，P>0.05。4W与12W时，BVF、BMD、弹性模量、反应力，Von Mises应力均升高P>0.01。并进行相关性比较，结果显示术后4周，弹性模量与Mankin评分相关系数
r=―0.835，（P<0.01）,有显著相关性，呈负相关。与骨密度相关系数r=0.848，（P<0.01）有显著相关性，呈正相关。与体积分数相关系数r=0.893，（P<0.01）,有显著相关性，呈正相关。12W时，弹性模量与Mankin评分相关系数r=―0.883，（P<0.01）,有显著相关性，呈负相关。与骨密度相关系数r=0.861，（P<0.01），有显著相关性，呈正相关；弹性模量与体积分数相关系数r=0.817，（P<0.01）,有显著相关性，呈正相关。
结论膝关节不稳早期软骨下骨发生力学性能改变，早期弹性模量明显降低，后期升高。同时弹性模量和软骨下骨的BMD、体积分数及关节软骨的Mankin评分密切相关。二磷酸盐可通过抗骨吸收明显提高软骨下骨的弹性模量，表明关节不稳早期软骨下骨力学性能的改变可能和异常应力引起的骨吸收有关。软骨下骨力学性能的改变在OA发生发展中具有重要作用。
第三部分兔膝骨关节炎早期软骨下骨血管再生变化及二磷酸盐干预效应的实验研究
目的观察兔膝关节不稳早期关节软骨改变和软骨下骨血管再生变化以及二磷酸盐的保护作用，探讨OA早期关节软骨改变和软骨下骨血管再生变化之间的关系以及软骨下骨在OA中的作用。
方法健康雄性新西兰大白兔60只，随机数字表法60只新西兰大白兔随机分成三组，模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。采用兔关节不稳模型（切断前交叉韧带）右膝造模。二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重，模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。分别切取保留关节面上下各2cm骨的手术侧膝关节，清除软组织，进行大体评分。通过对关节软骨表面形态观察及HE染色观察,进
行关节软骨损伤Mankin评分。同时对软骨下骨骨软骨连接处的血管密度进行计数。通过计算穿过骨软骨连接处的血管数目来确定血管密度，亦即接触或穿过潮线的血管数目。并进行统计学比较。
结果4W时模型组出现2例出现关节积液和滑膜增生，关节面糜烂、粗糙；8w时2例出现积液及滑膜增生，滑液轻度混浊，软骨改变集中在股骨内髁关节面，软骨软化，关节面发黄纤维化明显，有大的缝隙出现；12w时滑膜结节样增生，滑液混浊，有2例股骨内髁关节面出现了大的裂缝和溃疡，并且见到骨赘。统计学分析结果显示统计学分析结果显示4w时模型组与二磷酸盐组、对照组比较，已出现软骨退变（P<0.01），8、12w时退变进一步加重（P<0.01）。而12w退变明显比4W时严重，（P<0.01）。对照侧多为正常关节软骨表现，Mainkin评分0-1分。HE染色显示，模型组和二磷酸盐组关节软骨损伤Mankin评分在兔膝关节不稳4w时模型组与二磷酸盐组、对照组比较，已出现软骨退变（P<0.01），8、12w时退变进一步加重（P<0.01）。而12w退变明显比4w时严重，（P<0.01）。显示，股骨内外髁骨软骨交界区的血管侵犯在OA早期与对照组相比明显增加（P<0.01）,8w、12w和4w有明显差别（P<0.01），股骨外髁8w时明显增加（P<0.01），股骨内髁比股骨外髁在每个时间点有明显增多（P<0.01）。对股骨内外髁不同组之间的骨软骨连接区的血管侵犯的密度也进行了比较，结果显示，模型组与对照组有统计学意义（P<0.01），模型组与用药组明显差别(P<0.01）。
结论膝关节不稳早期关节软骨开始退变，软骨下骨与关节软骨交界区血管增生。二磷酸盐对关节软骨有明显的保护作用，其作用机制可能与其抑制血管增生，抗骨吸收改善关节不稳早期软骨下骨的力学性能有关。关节软骨和软骨下骨是一个相互联系的统一体，软骨下骨在OA的发病中可能有着更为重要的作用。
第四部分兔骨关节炎模型早期关节软骨及软骨下骨生化改变及二磷酸盐的干预效应
目的观察兔膝关节不稳早期关节软骨基质金属蛋白酶-13（MMP-13）、软骨下骨基质金属蛋白酶-9(MMP-9)和组织蛋白酶K(CK)的表达变化及二磷酸盐的抑制作用，进一步证明OA早期软骨下骨存在骨吸收，并探讨其机制及其二磷酸盐的作用途径。
方法健康雄性新西兰大白兔60只，随机数字表法60只新西兰大白兔随机分成三组，模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。采用兔关节不稳模型（切断前交叉韧带）右膝造模。二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重，模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。在各个时相点模型组和二磷酸盐组各取8个右膝关节，对照组取4个右膝关节的股骨内侧髁，用SP免疫组化染色方法分别检测各组在4W和12W时关节软骨MMP-13、软骨下骨MMP-9和CK表达变化，并进行统计学比较分析。
结果兔膝关节不稳造模4W时各组都有MMP-13、MMP-9和CK阳性表达细胞。对照组和二磷酸盐组阳性细胞较少，模型组阳性细胞较多。对照组、二磷酸盐组和模型组MMP-13阳性表达率分别为4.17%、10%和95%；对照组、二磷酸盐组和模型组MMP-9阳性表达率分别为4%、12.5%和90%；CK阳性表达率分别为8.33%、12.5%和90%。与对照组比较，模型组有显著性差异(P<0.01)，而二磷酸盐组无显著性差异(P>0.05)。二磷酸盐能明显减少MMP-13、MMP-9和CK阳性表达；与模型组比较，有显著性差异(P<0.01)。兔膝关节不稳造模8W时各组都有MMP-13、MMP-9和CK阳性表达细胞。对照组和二磷酸盐组阳性细胞较少，模型组阳性细胞较多。对照组、二磷酸盐组和模型组MMP-13阳性表达率分别为8.33%、12.5%和95%，对照组、二磷酸盐组和模型组MMP-9阳性表达率分别为8.33%、12.5%和87.5%；CK阳性表达率分别为8.33%、12.5%和85%。。与对照组比较，模型组有显著性差异(P<0.01)，而二磷酸盐组无显著性差异(P>0.05)。二磷酸盐能明显减少MMP-13、MMP-9和CK阳性表达；与模型组比较，有显著性差异(P<0.01)。兔膝关节不稳造模12W时各组都有MMP-13、MMP-9和CK阳性表达细胞。对照组和二磷酸盐组阳性细胞较少，模型组阳性细胞较多。对照组、二磷酸盐组和模型组MMP-13阳性表达率分别为8.0%、12.5%和77.5%，对照组、二磷酸盐组和模型组MMP-9阳性表达率分别为8.33%、15%和77.5%；CK阳性表达率分别为8.33%、12.5%和75%。。与对照组比较，模型组有显著性差异(P<0.01)，而二磷酸盐组无显著性差异(P>0.05)。二磷酸盐能明显减少MMP-13、MMP-9和CK阳性表达细胞；与模型组比较有显著性差异(P<0.01)。模型组MMP-9和CK阳性数目和阳性表达率在12W较4W时显著降低(P<0.05)。
结论膝关节不稳早期软骨下骨骨吸收明显， MMP-9和CK明显增高是其原因之一。随着OA的进展，骨吸收作用逐渐减弱。二磷酸盐的抗骨吸收作用与其抑制破骨细胞产生的MMP-9和CK有关。同时MMP-13的结果也看出二磷酸盐作用的多样性。
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 stage
Objective：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 stage
Objective： 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.
目的观察兔膝关节不稳早期软骨下骨三维结构变化及二磷酸盐的干预作用，以探讨骨性关节炎(OA)早期软骨下骨三维结构的变化在OA发生发展中的作用。
方法健康雄性新西兰大白兔60只，随机数字表法分为模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。。采用兔关节不稳模型（切断前交叉韧带）右膝造模。分三组：二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重；模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。分别切取保留关节面上下各2cm骨的手术侧膝关节，清除软组织，后行Micro-CT检查。得具体测量参数：骨体积分数(BVF),、骨小梁厚度Tb.Th)、骨小梁间隔(Tb.Sp)、骨小梁数量(Tb.N)、体积骨密度(vBMD)、组织骨密度（tBMD），并进行统计学分析。
结果模型组、二磷酸盐组、对照组不同时期的骨密度及软骨下骨微结构参数变化。显示术后第4W三组比较，模型组体积分数（BVF）、骨小梁数量(Tb.N)、骨小梁厚度（Tb.Th）比对照组明显降低，P<0.01；体积分数(BVF)比二磷酸盐组也降低P<0.05；BVF二磷酸盐组与对照组比亦降低,P<0.05；骨小梁分离度（Tb.Sp）模型组比二磷酸盐及对照组均增大,P<0.01。二磷酸盐组比对照组明显增大,P<0.01。骨密度模型组比二磷酸盐及对照组明显降低,P<0.05。而二磷酸盐组与对照组无统计学差异,P>0.05。12周时三组比较，模型组体积分数（BVF）、骨小梁厚度（Tb.Th）、骨小梁数目(Tb.N)比二磷酸盐及对照组明显增加,P<0.05。骨小梁分离度明显减少,P<0.05。而骨密度显著增加,P<0.01。我们对不同时期相关微结构参数也进行了比较,统计学分析。4周时和12周时软骨下骨微结构及骨密度变化情况比较，骨体积分数，骨小梁厚度，骨小梁数目明显增加,P<0.01。骨小梁分离度明显减小,P<0.01。12周时的骨密度较4周时显著增加,P<0.01。
结论膝关节不稳早期软骨下骨三维结构发生明显改变，表现明显的骨重塑。早期以骨破坏为主，各种骨量及骨结构指标均下降。后期出现明显的骨形成，各种骨量及骨结构指标均增加。二磷酸盐可通过抗骨吸收明显抑制软骨下骨的骨重塑，缓解骨质破坏和吸收，保护软骨下骨的骨结构，进而保护软骨下骨的力学性能，从而保护关节软骨。软骨下骨三维结构的改变在OA发生发展中具有重要作用。
第二部分骨关节炎软骨下骨力学性能变化有限元分析及二磷酸盐的干预效应
目的观察兔膝关节不稳早期软骨下骨力学性能的改变及二磷酸盐的干预作用，以探讨骨性关节炎(OA)早期软骨下骨力学性能的改变在OA发生发展中的作用。
方法健康雄性新西兰大白兔60只，随机数字表法60只新西兰大白兔随机分成三组，模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。采用兔关节不稳模型（切断前交叉韧带）右膝造模。二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重，模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。分别切取保留关节面上下各2cm骨的手术侧膝关节，清除软组织，进行大体评分。后行Micro-CT检查。从Micro-CT得到DICOM式的二维图像文件。将获得的DICOM导入Mimics V10.01软件中进行拟合，并导入到Ansys V10中进行有限元处理。得到关节软骨组织损伤Mankin评分；通过Micro-CT对60个标本进行检查，得到体积分数（BVF）、骨小梁厚度（Tb.Th）骨小梁数目(Tb.N)，骨小梁分离度(Tb.Sp)，骨密度（BMD）有限元软件计算得到弹性模量(EM)，反应力(RF)，平均Von Mises应力。后进行统计学分析比较。
结果兔膝关节不稳造模术后4W时，模型组、二磷酸盐组、对照组三组比较体积分数、弹性模量、反应力、平均Von Mises应力参数均下降，模型组比二磷酸盐组、对照组低, P<0.01。二磷酸盐组与对照组也下降，但P>0.05。骨密度模型组比二
磷酸盐及对照组明显降低，而二磷酸盐组与对照组无统计学差异（P>0.05）。12W时
模型组体积分数（BVF）、骨密度（BMD）明显增加, P<0.01。弹性模量、反应力，
Von Mises应力较对照组和二磷酸盐组低;二磷酸盐组亦较对照组低，但无统计意义，P>0.05。4W与12W时，BVF、BMD、弹性模量、反应力，Von Mises应力均升高P>0.01。并进行相关性比较，结果显示术后4周，弹性模量与Mankin评分相关系数
r=―0.835，（P<0.01）,有显著相关性，呈负相关。与骨密度相关系数r=0.848，（P<0.01）有显著相关性，呈正相关。与体积分数相关系数r=0.893，（P<0.01）,有显著相关性，呈正相关。12W时，弹性模量与Mankin评分相关系数r=―0.883，（P<0.01）,有显著相关性，呈负相关。与骨密度相关系数r=0.861，（P<0.01），有显著相关性，呈正相关；弹性模量与体积分数相关系数r=0.817，（P<0.01）,有显著相关性，呈正相关。
结论膝关节不稳早期软骨下骨发生力学性能改变，早期弹性模量明显降低，后期升高。同时弹性模量和软骨下骨的BMD、体积分数及关节软骨的Mankin评分密切相关。二磷酸盐可通过抗骨吸收明显提高软骨下骨的弹性模量，表明关节不稳早期软骨下骨力学性能的改变可能和异常应力引起的骨吸收有关。软骨下骨力学性能的改变在OA发生发展中具有重要作用。
第三部分兔膝骨关节炎早期软骨下骨血管再生变化及二磷酸盐干预效应的实验研究
目的观察兔膝关节不稳早期关节软骨改变和软骨下骨血管再生变化以及二磷酸盐的保护作用，探讨OA早期关节软骨改变和软骨下骨血管再生变化之间的关系以及软骨下骨在OA中的作用。
方法健康雄性新西兰大白兔60只，随机数字表法60只新西兰大白兔随机分成三组，模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。采用兔关节不稳模型（切断前交叉韧带）右膝造模。二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重，模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。分别切取保留关节面上下各2cm骨的手术侧膝关节，清除软组织，进行大体评分。通过对关节软骨表面形态观察及HE染色观察,进
行关节软骨损伤Mankin评分。同时对软骨下骨骨软骨连接处的血管密度进行计数。通过计算穿过骨软骨连接处的血管数目来确定血管密度，亦即接触或穿过潮线的血管数目。并进行统计学比较。
结果4W时模型组出现2例出现关节积液和滑膜增生，关节面糜烂、粗糙；8w时2例出现积液及滑膜增生，滑液轻度混浊，软骨改变集中在股骨内髁关节面，软骨软化，关节面发黄纤维化明显，有大的缝隙出现；12w时滑膜结节样增生，滑液混浊，有2例股骨内髁关节面出现了大的裂缝和溃疡，并且见到骨赘。统计学分析结果显示统计学分析结果显示4w时模型组与二磷酸盐组、对照组比较，已出现软骨退变（P<0.01），8、12w时退变进一步加重（P<0.01）。而12w退变明显比4W时严重，（P<0.01）。对照侧多为正常关节软骨表现，Mainkin评分0-1分。HE染色显示，模型组和二磷酸盐组关节软骨损伤Mankin评分在兔膝关节不稳4w时模型组与二磷酸盐组、对照组比较，已出现软骨退变（P<0.01），8、12w时退变进一步加重（P<0.01）。而12w退变明显比4w时严重，（P<0.01）。显示，股骨内外髁骨软骨交界区的血管侵犯在OA早期与对照组相比明显增加（P<0.01）,8w、12w和4w有明显差别（P<0.01），股骨外髁8w时明显增加（P<0.01），股骨内髁比股骨外髁在每个时间点有明显增多（P<0.01）。对股骨内外髁不同组之间的骨软骨连接区的血管侵犯的密度也进行了比较，结果显示，模型组与对照组有统计学意义（P<0.01），模型组与用药组明显差别(P<0.01）。
结论膝关节不稳早期关节软骨开始退变，软骨下骨与关节软骨交界区血管增生。二磷酸盐对关节软骨有明显的保护作用，其作用机制可能与其抑制血管增生，抗骨吸收改善关节不稳早期软骨下骨的力学性能有关。关节软骨和软骨下骨是一个相互联系的统一体，软骨下骨在OA的发病中可能有着更为重要的作用。
第四部分兔骨关节炎模型早期关节软骨及软骨下骨生化改变及二磷酸盐的干预效应
目的观察兔膝关节不稳早期关节软骨基质金属蛋白酶-13（MMP-13）、软骨下骨基质金属蛋白酶-9(MMP-9)和组织蛋白酶K(CK)的表达变化及二磷酸盐的抑制作用，进一步证明OA早期软骨下骨存在骨吸收，并探讨其机制及其二磷酸盐的作用途径。
方法健康雄性新西兰大白兔60只，随机数字表法60只新西兰大白兔随机分成三组，模型组（n=24）,二磷酸盐组（n=24）,对照组（n=12）。采用兔关节不稳模型（切断前交叉韧带）右膝造模。二磷酸盐组每天皮下注射二磷酸盐（利塞磷酸钠，Risedronate sodium）0.01mg/kg体重，模型组和对照组给予等体积的生理盐水皮下注射。术后分别于4W、8W、12W各时相点分别空气栓塞处死兔子，模型组（n=8），二磷酸盐组（n=8）,对照组（n=4）。在各个时相点模型组和二磷酸盐组各取8个右膝关节，对照组取4个右膝关节的股骨内侧髁，用SP免疫组化染色方法分别检测各组在4W和12W时关节软骨MMP-13、软骨下骨MMP-9和CK表达变化，并进行统计学比较分析。
结果兔膝关节不稳造模4W时各组都有MMP-13、MMP-9和CK阳性表达细胞。对照组和二磷酸盐组阳性细胞较少，模型组阳性细胞较多。对照组、二磷酸盐组和模型组MMP-13阳性表达率分别为4.17%、10%和95%；对照组、二磷酸盐组和模型组MMP-9阳性表达率分别为4%、12.5%和90%；CK阳性表达率分别为8.33%、12.5%和90%。与对照组比较，模型组有显著性差异(P<0.01)，而二磷酸盐组无显著性差异(P>0.05)。二磷酸盐能明显减少MMP-13、MMP-9和CK阳性表达；与模型组比较，有显著性差异(P<0.01)。兔膝关节不稳造模8W时各组都有MMP-13、MMP-9和CK阳性表达细胞。对照组和二磷酸盐组阳性细胞较少，模型组阳性细胞较多。对照组、二磷酸盐组和模型组MMP-13阳性表达率分别为8.33%、12.5%和95%，对照组、二磷酸盐组和模型组MMP-9阳性表达率分别为8.33%、12.5%和87.5%；CK阳性表达率分别为8.33%、12.5%和85%。。与对照组比较，模型组有显著性差异(P<0.01)，而二磷酸盐组无显著性差异(P>0.05)。二磷酸盐能明显减少MMP-13、MMP-9和CK阳性表达；与模型组比较，有显著性差异(P<0.01)。兔膝关节不稳造模12W时各组都有MMP-13、MMP-9和CK阳性表达细胞。对照组和二磷酸盐组阳性细胞较少，模型组阳性细胞较多。对照组、二磷酸盐组和模型组MMP-13阳性表达率分别为8.0%、12.5%和77.5%，对照组、二磷酸盐组和模型组MMP-9阳性表达率分别为8.33%、15%和77.5%；CK阳性表达率分别为8.33%、12.5%和75%。。与对照组比较，模型组有显著性差异(P<0.01)，而二磷酸盐组无显著性差异(P>0.05)。二磷酸盐能明显减少MMP-13、MMP-9和CK阳性表达细胞；与模型组比较有显著性差异(P<0.01)。模型组MMP-9和CK阳性数目和阳性表达率在12W较4W时显著降低(P<0.05)。
结论膝关节不稳早期软骨下骨骨吸收明显， MMP-9和CK明显增高是其原因之一。随着OA的进展，骨吸收作用逐渐减弱。二磷酸盐的抗骨吸收作用与其抑制破骨细胞产生的MMP-9和CK有关。同时MMP-13的结果也看出二磷酸盐作用的多样性。
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 stage
Objective：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 stage
Objective： 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.
引文
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4、Janet M, Helen L, Roger L, et al. Fundamental subchondral bone changes in spontane-ousknee osteoarthritis. Cell Biology,2005,37:224-236.
5、Hayami T，Pickarski M，Zhuo Y，et a1．Characterization Of articular cartilage andsubchondral bone changes in the rat anterior cruclate ligament transection andmeniscectomized models of osteoarthfitis[J]．Bone，2006，38(2)：234—243.
6、Goldring MB，Goldring SR．Articular cartilage and subchondral bone in thepathogenesis of osteoarthritis．Ann N Y Acad Sci，2010，1192：230—237．
7、P. I. Mapp Ph.D., P. S. Avery B.Sc., D. F. McWilliams Ph.D., Angiogenesis in twoanimal models of osteoarthritis. Osteoarthritis and Cartilage (2008)16,61-69.
8、David A. Walsh, Dan F. McWilliams, Matthew J. Turley, Angiogenesis and nervegrowth factor at the osteochondral junction in rheumatoid arthritis and osteoarthritis.Rheumatology2010;49:1852–1861.
9、Brandt KD, Doherty M, Lohmander LS. Osteoarthritis. London, UK: Oxford UniversityPress;1998.
10、Paul I. Mapp and David A. Walsh, Mechanisms and targets of angiogenesis andnerve growth in osteoarthritis. Rheumatoloy,2012,8:390-398
11、M. Saito y, T. Sasho y*, S. Yamaguchi y, N. Angiogenic activity of subchondral boneduring the progression of osteoarthritis in a rabbit anterior cruciate ligament transectionmodel. Osteoarthritis and Cartilage1220,(20),1574-1582.
12、Massicotte F, Martel–Pelletier J, Pelletier J-P, et al.: Abnormal modulation ofinsulin-like growth factor1levels in human osteoarthritic bone osteoblasts [abstract].Arthritis Rheum,2000,43:S206.
13、Blanquaert F, Pereira RC, Canalis E: Cortisol inhibits hepatocyte growth factor/scatterfactor expression and induces c-met transcripts in osteoblasts. Am J Physiol EndocrinolMetab,2000,278:E509–E515.
1、Pelletier JP, Martel-Pelletier J, Howell DS. Etiopathogenesis of osteoarthritis. ATextbook of Rheumatology，2001, p:2195-245(chapter110).
2、Feuerherm AJ, Borset M, Seidel C, et al.: Elevated levels of osteoprotegerin (OPG) andhepatocyte growth factor (HGF) in rheumatoid arthritis. Scand J Rheumatol,2001,30:229–234.
3、Hayami T，Pickarski M，Zhuo Y，et a1．Characterization Of articular cartilage andsubchondral bone changes in the rat anterior cruclate ligament transection andmeniscectomized models of osteoarthfitis[J]．Bone，2006，38(2)：234—243.
4、Lajeunesse D, Martel-Pelletier J, Fernandes JC,et al.Treatment with licofelone preventsabnormal subchondral bone cell metabolism in experimental dog osteoarthritis. AnnRheum Dis,2004,63:78–83.
5、谢希，高杰生.骨关节炎动物模型研究进展[J].医学综述2005,11(1)：67-69
6、Brandt KD, Doherty M, Lohmander LS. Osteoarthritis. London, UK: Oxford UniversityPress;1998.
7、Sakao K，Takahashi KA，Mazda0，et a1．Enhanced expression of interleukin--6，matrix metallopreteinase-13， and receptor activator of NF—kappaB ligand incellsderived from osteoarthritic subchondral bone. J Orthop sci,2008，13(3)：202-210.
8、Kwan Tat S，Pelletier JP，Lajeunesse D，et a1．The di erential expression ofosteo-protegerin(0PG) and receptor activator of nuclear factor kappaB ligand(RANKL)inhuman osteoarthritic subchondral bone osteoblasts is all indicator of the metabolicstate of these disease cells．Clin Exp Rheumatol，2008,26（2）：295-304
9、王玉彬，陈安民，郭风劲等.基质金属蛋白酶家族在骨关节炎软骨组织中表达的研究.中国矫形外科杂志,2007,15(11):853-855.
10、周辉，董刚，夏志敏等. MMP．1、MMP一9mRNA在创伤性骨关节炎软骨中的表达.中国骨质疏松杂志，2009，15（2）96-98
11、魏巍，孙文靖，金焰等.人组织蛋白酶K基因研究进展.国际遗传学杂志，2012，35（2）82-85.
12、Selingerc I，Day CJ，Morrision NA，et a1．Optimized transfection of diced siRNAinto mature primary human osteoclasts：inhibition of cathepsin k mediated boneresorption by siRNA. J Cell Biochem，2005(96)：996-1002．
13、Stroup G B，Lark M W，Veber D F，et a1．Potent and selective inhibition of humancathepsinK leads to inhibition of bone resorption in vivo in a nonhuman primate[J]．JBone Miner Res，2001，16：1739—1746．
14、Wittrant Y，Couillaud S，Theoleyrd S，et a1．Osteoprotegerin differentially regulatesprotease expression in osteoclast cultures[,J]．J Biochem Biophys Res Commun，2002，293：38—44．
15、史晓林,刘康,吴连国.骨碎补总黄酮对骨质疏松靶标一组织蛋白酶K干预价值的探讨. Chinese Trad Med Traum&Orthop．2008，16(5):70-72.
16、王运林，刘晓睛，杨菲等.金雀异黄素抑制IL.1a刺激破骨样细胞的组织蛋白酶K表达.Chinese Journal of Cell Biology2006．28：473—476.
1、Li W，Abram F．Human hip joint cartilage：MRI quantitative thickness and volumemeasurements discriminating acetabulum and femoral head[J]．IEEE Trans BiomedEng，2008，55(12)：2731—2740．
2、杨玉海，崔谊，崔允峰等．螺旋CT三维重建评价膝关节创伤的临床应用价值．医学影像学杂志，2004，14(1)：45
3、Ben djaballah MZ，Shirazi—Adl A，Zukor DJ．Finite dement analysis of human kneejoint in varus—valgus．Clin Biomech (Bristol，Avon)，1997，12(3)：139
4、伍中庆，吴宇峰，苏培基等．膝关节三维有限元模型的建立．中华实用中西医杂志，2004，4(17)：2970-71
5、汪强，孙俊英，赖震等．膝关节三维有限元模型的建立．陕西医药杂志，2007,36（3）：210-212
6、Hirokawa，S．，Tsuruno，R．，Three-dimensional deformation and stress distributionin an analytical／computational modelof the anterior cruciate ligament[J]．Journal ofBiomechanics，2000，33：1069-1077．
7、王光达，张祚福，齐晓军等.膝关节三维有限元模型的建立及生物力学分析.中国组织工程研究与临床康复，2010,14（52）：9702-9705
8、黄建国，史庆南，严继康等。膝关节三维有限元模型的重建。中华医学研室杂志，2006，6（4）：415-416
9、姜华亮，华锦明，许新忠等．正常人膝关节三维有限元模型的建立。苏州大学学报(医学版),2008；28(3)：421-422
10、G Limbert，M．Taylor，J．Middleton．Three-dimensional finite element modelingof the human ACL：simulation of passive knee flexion with a stressed and stress-freeACL [J]．Journal of Biomechanics，2004，37：1723-1731．
11、姚杰，牛文鑫，王旸等．跳伞着陆过程中膝关节损伤的有限元研究.医用生物力学，2010，25（4）：244-248
12、吴宇峰，苏培基，伍中庆等．髌骨的三维有限元重建及初步力学分析.中国中医骨伤科杂志,2004,12(2):1-3
13、辛力，王业华.合并膝关节脱位的胫骨内侧平台骨折4种内固定方法的生物力学性能静态有限元分析.徐州医学院学报,2008,28(8):533-536
14、Bougherara H, Zdero R, Mahboob Z，et a1．The biomechanics of a validated finiteelement model of stress shielding in a novel hybrid total knee replacement. Proc Inst Mech Eng H.2010Oct;224(10):1209-19.
15、Completo A, Rego A, Fonseca F, et a1．Biomechanical evaluation of proximal tibia behaviourwith the use of femoral stems in revision TKA: an in vitro and finite element analysis. ClinBiomech (Bristol, Avon).2010Feb;25(2):159-65.
16、liau J J，Cheng C K，Huang C H，et a1．The efect of malalignment on stresses inpolyethylene component of total knee prostheses-a finite element analysis．ClinicalBiomechanics，2002，17：140-146
17、Completo A, Sim es JA, Fonseca F. Revision total knee arthroplasty: the influence of femoralstems in load sharing and stability. Knee.2009Aug;16(4):275-9.
18、Chandran N, Amirouche F, Gonzalez MH, et a1．Optimisation of the posterior stabilisedtibial post for greater femoral rollback after total kneearthroplasty--a finite element analysis. IntOrthop.2009Jun;33(3):687-93.
2. Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease:potential implication for the selection of new therapeutic targets. Arthritis Rheum,2001,44:1237-47.
3、Clouet J，Vinatier C，Merceron C，et a1．From osteoarthritis treatments to futureregenerative therapies for cartilage．Drug Discov Today，2009，14(19—2l)：913—925．
4、Janet M, Helen L, Roger L, et al. Fundamental subchondral bone changes in spontane-ousknee osteoarthritis. Cell Biology,2005,37:224-236.
5、Brandt KD, Doherty M, Lohmander LS. Osteoarthritis. London, UK: Oxford UniversityPress;1998.
6、Marcu KB，Otero M，Olivotto E，et a1．NF—kappaB signaling：multiple angles totarget OA．Curr Drug Targets，2010，11(5)：599—613．
7、Radin EL，Rose RM．Role of subchondra1bone in the initiation and progression ofcartilage damage．Clin Orthop Relat Res,1986，(213)：34—40．
8、 Goldring MB， Goldring SR． Articular cartilage and subchondral bone in thepathogenesis of osteoarthritis．Ann N Y Acad Sci，2010，1192：230—237．
9、 Kwan Tat S，Lajeunesse D，Pelletier JP，et a1．Targeting subchondral bone for treatingosteoarthritis：what is the evidence? Best Pract Res Clin Rheurnato1．2010，24(1)：51—70．
10、Karsdal MA，Leeming DJ，Dam EB，et al．Should subchondral bone turnover betargeted wllen treating osteoarthritis? Osteoarthritis Cartilage，2008，16(6)：638—646．
11、Lajeunesse D, Martel-Pelletier J, Fernandes JC, et al.Treatment with licofeloneprevents abnormal subchondral bone cell metabolism in experimental dog osteoarthritis.Ann Rheum Dis,2004,63:78–83.
12、McKinley TO, Bay BK. Trabecular bone strain changes associated with subchondralstiffening of the proximal tibia. J Biomech,2003,36:155-63.
13、Bailey AJ, Sims TJ, Knott L: Phenotypic expression of osteoblast collagen inosteoarthritic bone: production of type I homotrimer. Int J Biochem Cell Biol,2002,34:176–182.
14、Blaney Davidson EN，van der Kraan PM, van den Berg WB．TGF-beta and osteoarthritis．Osteoarthritis Cartilage，2007，15(6)：597—604．
15、Okazaki R, Sakai A, Uezono Y, et a1. Sequential changes in transforming growthfactor(TGF)-betal concentration in synovial fluid and mRNA expression of TGF-betalreceptors in chondroc ytes after immobililization of rabbit knees[J]. J Bone MinerMetab,2001,19(4):228-35.
16、 Burr DB. The importance of subchondral bone in osteoarthrosis. Curr OpinRheumatol,1998,10:256-562.
1、Clouet J，Vinatier C，Merceron C，et a1．From osteoarthritis treatments to futureregenerative therapies for cartilage．Drug Discov Today，2009，14(19—2l)：913—925．
2、Danika L,Batiste M.Sc., Alexandra Kirkley et a1．Ex vivo characterization of articularcartilage and bone lesions in a rabbit ACL transection model of osteoarthritis usingMRI and micro-CT1OsteoArthritis and Cartilage (2004)12,986-996.
3、Ming Ding, Carl Christian Danielsen, Ivan Hvid. The Effects of Bone RemodelingInhibition by Alendronate on Three-Dimensional Microarchitecture of SubchondralBone Tissues in Guinea Pig Primary Osteoarthrosis. Calcif Tissue Int (2008)82:77–86.
4、Janet M, Helen L, Roger L, et al. Fundamental subchondral bone changes in spontane-ousknee osteoarthritis. Cell Biology,2005,37:224-236.
5、Bettica P, Cline G, Hart DJ, et al. Evidence for increased bone resorption in patientswith progressive knee osteoarthritis: longitudinal results from the Chingford study.Arthritis Rheum,2002,46:3178-84.
6、Joshua A, MacNeil, Steven K. Boyd. Bone strength at the distal radius can be estimatedfrom high-resolution peripheral quantitative computed tomography and the finiteelement method. Bone42(2008)1203–1213
7、王军，毕龙，白建萍，等．显微CT与组织切片技术在骨形态计量研究中的比较[J]．中国矫形外科杂志，2009，l7(5)：38l一384．
8、Chappard D, Retailleau--Gaborit N, Legrand E, Audran M. Comparison Insight BoneMeasurement s by Histomorphometry and CT.Journal of Bone and MineralResearch,2005;20(7)∶1177
9、Feldkamp LA， Goldstein SA，Parfitt AM，et a1．The direct examination ofthree—dimensional bone architecture in vitro by computed tomography．J Bone MinerRes．1989．4：3-11．
10、Chapurlat RD, Arlot M, Burt-Pichat B, Chavassieux P, Roux JP, Portero-Muzy N,Delmas PD. Microcrack frequency and bone remodeling in postmenopausalosteoporotic women on long-term bisphosphonates: a bone biopsy study. J BoneMiner Res2007;22:1502–9.
11、Nehme A，Maalouf G，Tricoire J I，et a1．Effect of alendronate on periprosthetic boneloss after cemented primary total hip arthroplasty：prospective randomized study[J]Rev Chir Orthop Repatatrice Appar Mot，2003，89(7)：593—598．
12、Iwamoto J, Takeda T, Sato Y, Matsumoto H. Effects of risedronate on osteoarthritis ofthe knee. Yonsei Med J.2010Mar;51(2):164-70.
13、Joshua A. MacNeil, Michael R. Doschak, Ronald F. Zernicke. Preservation ofperiarticular cancellous morphology and mechanical stiffness in post-traumaticexperimental osteoarthritis by antiresorptive therapy. Clinical Biomechanics23(2008)365–371
1、Janet M, Helen L, Roger L, et al. Fundamental subchondral bone changes inspontaneous knee osteoarthritis. Cell Biology,2005,37:224-236.
2、Blaney Davidson EN，van der Kraan PM, van den Berg WB．TGF-beta and osteoarthritis．Osteoarthritis Cartilage，2007，15(6)：597—604．
3、Sakao K, Takahashi KA, Arai Y, Saito M, Honjyo K. Asporin and transforming growthfactor-beta gene expression in osteoblasts from subchondral bone and osteophytes inosteoarthritis. J Orthop Sci.2009Nov;14(6):738-47.
4、Ahmed S, Silverman MD, Marotte H. Down-regulation of myeloid cell leukemia1byepigallocatechin-3-gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumornecrosis factor alpha-induced apoptosis.Arthritis Rheum.2009May;60(5):1282-93.
5、Homminga J, Huiskes R, Van Rietbergen B, Ruegsegger P, Weinans H.Introduction andevaluation of a gray-value voxel conversion technique. J Biomech2001;34:513–7.
6、Schencking M, Wilm S, Redaelli M. A comparison of Kneipp hydrotherapy withconventional physiotherapy in the treatment of osteoarthritis: a pilot trial. J Integr Med.2013Jan;11(1):17-25.
7、Teo JC，Si—Hoe KM，Keh JE，et a1．Relationship between CT intensity，micro-architecture and mechanical properties of porcine vertebral cancellousbone[J]．Clin Biomech，2006，21(3)：235—244．
8、McKinley TO, Borrelli J Jr, D'Lima DD, et a1．Basic science of intra-articular fracturesand posttraumatic osteoarthritis. J Orthop Trauma.2010Sep;24(9):567-70.
9、董启榕，陈向阳．骨性关节炎软骨下骨力学性能变化的实验研究．中华创伤杂志，2007，23(5)：386-389．
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11、Fazzalari NL, Kuliwaba JS, Forwood MR. Cancellous bone microdamage in theproximal femur: influence of age and osteoarthritis on damage morphology andregional distribution. Bone.2002Dec;31(6):697-702.
12、Radin EL，Rose RM．Role of subchondra1bone in the initiation and progression ofcartilage damage．Clin Orthop Relat Res,1986，(213)：34—40．
13、Ebetino F, Bayless A, Amburgey J, et a1.Elucidation of a pharmacophore for thebisphosphol1ate mechanism of bone resorptive activity. Phosphorus Sulfur Silicon,1996,109(10)：217-20．
14、MacNeil, J.A., Boyd, S.K.,2007. Bone fracture strength finite element measurementsusing in vivo high resolution peripheral quantitative computed tomography. In:Proceedings of the21st Congress of the International Society of Biomechanics,Springer, Taiwan.
1、Pelletier JP, Martel-Pelletier J, Howell DS. Etiopathogenesis of osteoarthritis. ATextbook of Rheumatology，2001, p:2195-245(chapter110).
2、BLom A B，van der Kraan P M，van den Berg W B. Cytokine targeting inosteoathritis[J]．Curr Drug Targets，2007，8(2)：283—292．
3、D. A. Walsh F.R.C.P., C. S. Bonnet B.Sc., E. L. Turner B.Sc., Angiogenesis in thesynovium and at the osteochondral junction in Osteoarthritis. Osteoarthritis andCartilage (2007)15,743-751.
4、Janet M, Helen L, Roger L, et al. Fundamental subchondral bone changes in spontane-ousknee osteoarthritis. Cell Biology,2005,37:224-236.
5、Hayami T，Pickarski M，Zhuo Y，et a1．Characterization Of articular cartilage andsubchondral bone changes in the rat anterior cruclate ligament transection andmeniscectomized models of osteoarthfitis[J]．Bone，2006，38(2)：234—243.
6、Goldring MB，Goldring SR．Articular cartilage and subchondral bone in thepathogenesis of osteoarthritis．Ann N Y Acad Sci，2010，1192：230—237．
7、P. I. Mapp Ph.D., P. S. Avery B.Sc., D. F. McWilliams Ph.D., Angiogenesis in twoanimal models of osteoarthritis. Osteoarthritis and Cartilage (2008)16,61-69.
8、David A. Walsh, Dan F. McWilliams, Matthew J. Turley, Angiogenesis and nervegrowth factor at the osteochondral junction in rheumatoid arthritis and osteoarthritis.Rheumatology2010;49:1852–1861.
9、Brandt KD, Doherty M, Lohmander LS. Osteoarthritis. London, UK: Oxford UniversityPress;1998.
10、Paul I. Mapp and David A. Walsh, Mechanisms and targets of angiogenesis andnerve growth in osteoarthritis. Rheumatoloy,2012,8:390-398
11、M. Saito y, T. Sasho y*, S. Yamaguchi y, N. Angiogenic activity of subchondral boneduring the progression of osteoarthritis in a rabbit anterior cruciate ligament transectionmodel. Osteoarthritis and Cartilage1220,(20),1574-1582.
12、Massicotte F, Martel–Pelletier J, Pelletier J-P, et al.: Abnormal modulation ofinsulin-like growth factor1levels in human osteoarthritic bone osteoblasts [abstract].Arthritis Rheum,2000,43:S206.
13、Blanquaert F, Pereira RC, Canalis E: Cortisol inhibits hepatocyte growth factor/scatterfactor expression and induces c-met transcripts in osteoblasts. Am J Physiol EndocrinolMetab,2000,278:E509–E515.
1、Pelletier JP, Martel-Pelletier J, Howell DS. Etiopathogenesis of osteoarthritis. ATextbook of Rheumatology，2001, p:2195-245(chapter110).
2、Feuerherm AJ, Borset M, Seidel C, et al.: Elevated levels of osteoprotegerin (OPG) andhepatocyte growth factor (HGF) in rheumatoid arthritis. Scand J Rheumatol,2001,30:229–234.
3、Hayami T，Pickarski M，Zhuo Y，et a1．Characterization Of articular cartilage andsubchondral bone changes in the rat anterior cruclate ligament transection andmeniscectomized models of osteoarthfitis[J]．Bone，2006，38(2)：234—243.
4、Lajeunesse D, Martel-Pelletier J, Fernandes JC,et al.Treatment with licofelone preventsabnormal subchondral bone cell metabolism in experimental dog osteoarthritis. AnnRheum Dis,2004,63:78–83.
5、谢希，高杰生.骨关节炎动物模型研究进展[J].医学综述2005,11(1)：67-69
6、Brandt KD, Doherty M, Lohmander LS. Osteoarthritis. London, UK: Oxford UniversityPress;1998.
7、Sakao K，Takahashi KA，Mazda0，et a1．Enhanced expression of interleukin--6，matrix metallopreteinase-13， and receptor activator of NF—kappaB ligand incellsderived from osteoarthritic subchondral bone. J Orthop sci,2008，13(3)：202-210.
8、Kwan Tat S，Pelletier JP，Lajeunesse D，et a1．The di erential expression ofosteo-protegerin(0PG) and receptor activator of nuclear factor kappaB ligand(RANKL)inhuman osteoarthritic subchondral bone osteoblasts is all indicator of the metabolicstate of these disease cells．Clin Exp Rheumatol，2008,26（2）：295-304
9、王玉彬，陈安民，郭风劲等.基质金属蛋白酶家族在骨关节炎软骨组织中表达的研究.中国矫形外科杂志,2007,15(11):853-855.
10、周辉，董刚，夏志敏等. MMP．1、MMP一9mRNA在创伤性骨关节炎软骨中的表达.中国骨质疏松杂志，2009，15（2）96-98
11、魏巍，孙文靖，金焰等.人组织蛋白酶K基因研究进展.国际遗传学杂志，2012，35（2）82-85.
12、Selingerc I，Day CJ，Morrision NA，et a1．Optimized transfection of diced siRNAinto mature primary human osteoclasts：inhibition of cathepsin k mediated boneresorption by siRNA. J Cell Biochem，2005(96)：996-1002．
13、Stroup G B，Lark M W，Veber D F，et a1．Potent and selective inhibition of humancathepsinK leads to inhibition of bone resorption in vivo in a nonhuman primate[J]．JBone Miner Res，2001，16：1739—1746．
14、Wittrant Y，Couillaud S，Theoleyrd S，et a1．Osteoprotegerin differentially regulatesprotease expression in osteoclast cultures[,J]．J Biochem Biophys Res Commun，2002，293：38—44．
15、史晓林,刘康,吴连国.骨碎补总黄酮对骨质疏松靶标一组织蛋白酶K干预价值的探讨. Chinese Trad Med Traum&Orthop．2008，16(5):70-72.
16、王运林，刘晓睛，杨菲等.金雀异黄素抑制IL.1a刺激破骨样细胞的组织蛋白酶K表达.Chinese Journal of Cell Biology2006．28：473—476.
1、Li W，Abram F．Human hip joint cartilage：MRI quantitative thickness and volumemeasurements discriminating acetabulum and femoral head[J]．IEEE Trans BiomedEng，2008，55(12)：2731—2740．
2、杨玉海，崔谊，崔允峰等．螺旋CT三维重建评价膝关节创伤的临床应用价值．医学影像学杂志，2004，14(1)：45
3、Ben djaballah MZ，Shirazi—Adl A，Zukor DJ．Finite dement analysis of human kneejoint in varus—valgus．Clin Biomech (Bristol，Avon)，1997，12(3)：139
4、伍中庆，吴宇峰，苏培基等．膝关节三维有限元模型的建立．中华实用中西医杂志，2004，4(17)：2970-71
5、汪强，孙俊英，赖震等．膝关节三维有限元模型的建立．陕西医药杂志，2007,36（3）：210-212
6、Hirokawa，S．，Tsuruno，R．，Three-dimensional deformation and stress distributionin an analytical／computational modelof the anterior cruciate ligament[J]．Journal ofBiomechanics，2000，33：1069-1077．
7、王光达，张祚福，齐晓军等.膝关节三维有限元模型的建立及生物力学分析.中国组织工程研究与临床康复，2010,14（52）：9702-9705
8、黄建国，史庆南，严继康等。膝关节三维有限元模型的重建。中华医学研室杂志，2006，6（4）：415-416
9、姜华亮，华锦明，许新忠等．正常人膝关节三维有限元模型的建立。苏州大学学报(医学版),2008；28(3)：421-422
10、G Limbert，M．Taylor，J．Middleton．Three-dimensional finite element modelingof the human ACL：simulation of passive knee flexion with a stressed and stress-freeACL [J]．Journal of Biomechanics，2004，37：1723-1731．
11、姚杰，牛文鑫，王旸等．跳伞着陆过程中膝关节损伤的有限元研究.医用生物力学，2010，25（4）：244-248
12、吴宇峰，苏培基，伍中庆等．髌骨的三维有限元重建及初步力学分析.中国中医骨伤科杂志,2004,12(2):1-3
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