| Part One Experimental research of establishing osteoarthritis model induced by anterior cruciate ligament transection combined with ovariectomy in ratsObjective: The aim of this study was to evaluate the effectiveness of establishing a model of osteoarthritis(OA)by ovariectomy(OVX)combined with anterior cruciate ligament transection(ACLT)in rats,and to provide an ideal animal model for basic research of postmenopausal OA.Methods:1.48 twelve-week-old female Sprague-Dawley rats were randomly divided into four groups of 12 rats each as follows: The sham group,ACLT group,OVX group,and ACLT + OVX group.Then,each group was randomly divided into two subgroups;one subgroup was used for histopathological analysis(n = 6)and another one was used for imaging evaluation(n = 6).The OVX and ACLT groups accepted bilateral OVX and ACLT on the right knee,respectively.The ACLT + OVX group received both OVX and ACLT,and the sham group was underwent only sham operation.2.After 8 weeks,the specimens for imaging analysis were disarticulated and imaged for macroscopic scoring.Then,the specimens were fixed in 75%ethanol for micro-computed tomography(micro-CT)analysis.The samples for pathological evaluation were fixed,decalcified,embedded,and sectioned.The sections were stained with toluidine blue and pathological evaluation was performed based on the scoring system of the Osteoarthritis Research Society International(OARSI).Results:1.The articular surface in the sham group was smooth and glossy,and the structure of articular capsule was normal.In the OVX group,the articular surface was basically smooth but slightly dark.In the ACLT group,there were superficial lesions in the cartilage surface of the articular surface accompanied by poor gloss and thickening of the articular capsule.However,serious erosions reaching the deep layer of the cartilage,a gray white remnant surface with small ulcers,and serious thickening of the articular capsule were found in the ACLT + OVX group.The macroscopic score in the ACLT group was higher than that in the sham group(P < 0.005)but lower than that in the ACLT + OVX group(P < 0.005).No significant difference was found between the OVX and sham groups(P > 0.05).2.According to the toluidine blue staining,a smooth articular cartilage surface was shown in the sham group,accompanied by the clear structure and layers,orderly arranged chondrocytes,evenly stained extracellular matrix(ECM),clear structure of tidemark and normal meniscus.In the OVX group,mild wrinkles in the cartilage surface and a little bit of fade in the ECM were observed.Superficial cartilage fibrillations,cracks deep into the middle layer of the cartilage,a decreased number of chondrocytes,obvious fade in the ECM,the fuzzy tidemark structure and fragmented meniscus were shown in the ACLT group.However,extensive erosions and cracks reaching the deep layer of the cartilage,a local subsidence of the subchondral bone,a significant reduction of chondrocytes,a wide range of fade in the ECM,unclear tidemark structure,and the disintegrated and displaced meniscus were detected in the ACLT + OVX group.The OARSI score in the ACLT group was significantly higher than that of the sham group(P < 0.005),but lower than that of the ACLT + OVX group(P < 0.005).There was no significant difference between the OVX and sham groups(P > 0.05).3.The micro-CT analysis showed that lower Tb.Th and higher SMI(all P< 0.05)were detected in the ACLT group compared with those in the sham group.BMD,BV/TV,and Tb.Th were significantly decreased and SMI was substantially increased in the OVX group compared with those in the sham group(all P < 0.05).However,BMD,BV/TV,and Tb.N were strongly decreased,and SMI and Tb.Sp were increased in the ACLT + OVX group compared with those in other three groups,respectively(all P < 0.05).Tb.Th in the ACLT + OVX group was markedly lower than that of the sham group(P < 0.001).Part Two Preventive and therapeutic effects of raloxifene on osteoarthritis induced by anterior cruciate ligament transection combined with ovariectomy in ratsObjective: The aim of this study was to investigate the preventive and therapeutic effects of raloxifene(RAL)on osteoarthritis(OA)induced by a combination of ovitectomy(OVX)and anterior cruciate ligament transection(ACLT)in rats.Methods:1.60 twelve-week-old female Sprague-Dawley rats were randomly divided into five groups of 12 rats each as follows: The sham group,ACLT group,OVX group,ACLT + OVX group,and RAL group(ACLT + OVX+RAL).Then,each group was randomly divided into two subgroups;one subgroup was used for histopathological analysis(n = 6)and another subgroup was used for imaging evaluation(n = 6).2.Bilateral OVX and ACLT on the right knee were performed in the OVX group and the ACLT group,respectively.The ACLT + OVX and RAL groups received both OVX and ACLT,and the sham group was underwent only sham operation.RAL was administered to the animals in the RAL group by oral gavage(6.25 mg/kg/day)daily at 72 hours after the surgery for 12 weeks.Other groups received the same volume of distilled water as a vehicle.3.After sacrifice,the right knees for imaging analysis were disarticulated and imaged for macroscopic scoring.Then,the specimens were fixed in 75%ethanol for micro-computed tomography(micro-CT)analysis.The samples for pathological evaluation were fixed,decalcified,embedded,and sectioned,sequentially.The sections were stained with toluidine blue and pathological evaluation was performed based on the scoring system of the Osteoarthritis Research Society International(OARSI).The protein levels of type II collagen(col-II)and aggrecan(AGG)in the cartilage were evaluated with integratal optical density(IOD)by immunohistochemistry.Results:1.The smooth and glossy articular surface and normal articular capsule were found in the sham group.The articular surface was basically smooth but dim in the OVX group.Obvious lesions reaching the middle layer of the cartilage,discoloration,and thickening of the articular capsule were shown in the ACLT group.However,deteriorated lesions,cartilage proliferation,subchondral bone exposure,and serious thickening of the articular capsule were detected in the ACLT + OVX group.Cartilage degeneration and gloss loss were all alleviated by RAL compared with the ACLT + OVX group.The macroscopic score in the ACLT group was higher than that in the sham group(P < 0.005)but lower than that in the ACLT + OVX group(P < 0.005).The score in the RAL group was markedly lower than that in the ACLT + OVX group(P < 0.005).No significant difference was detected between the OVX and sham groups(P > 0.05).2.Toluidine blue staining showed that a smooth cartilage surface,normal structure and layers,the orderly arranged chondrocytes,the evenly stained extracellular matrix(ECM),the clear tidemark structure and normal meniscus were shown in the sham group.Mild wrinkles in the cartilage surface and a slight decrease in the ECM staining were observed in the OVX group.Lesions reaching the middle layer of the cartilage,cracks into the deep layer,a significant reduction in the number of chondrocytes,an obvious decrease in the ECM staining,the fuzzy tidemark structure and cracked meniscus were showed in the ACLT group.However,extensive cartilage defects,subchondral bone exposure and defect,and the breakage of meniscus were detected in the ACLT + OVX group.Slighter degree of cartilage erosions,clearer structure of tidemark,better morphology of the subchondral bone and meniscus were found in the RAL group in comparison with those in the ACLT+ OVX group.The OARSI score in the ACLT group was significantly higher than that of the sham group(P < 0.005),but lower than that of the ACLT +OVX group(P < 0.005).The OARSI score in the RAL group was markedly lower than that in the ACLT + OVX group(P < 0.001).There was no significant difference between the OVX and sham groups(P > 0.05).3.Immunohistochemical result showed that the levels of Col-II(P <0.005)and AGG(P < 0.05)in the ACLT group were lower than those in the sham group.Significantly decreased levels of Col-II(P < 0.05)and AGG(P <0.05)were observed in the ACLT + OVX group compared with those in the ACLT group.As expected,RAL increased the levels of Col-II(P < 0.001)and AGG(P < 0.05)compared with those in the ACLT + OVX group.The expression levels of Col-II and AGG in the OVX group were lower than those in the sham group,but no significant difference was detected(all P > 0.05).4.Lower Tb.Th(P < 0.05)and higher SMI(P < 0.005)were detected in the ACLT group compared with those in the sham group.BMD(P < 0.01),BV/TV(P < 0.005),and Tb.Th(P < 0.05)were significantly decreased and SMI(P < 0.001)was substantially increased in the OVX group compared with those in the sham group.However,BMD,BV/TV,Tb.Th,and Tb.N were strongly decreased,and SMI and Tb.Sp were increased in the ACLT + OVX group compared with those in the ACLT and the OVX groups,respectively(all P < 0.05).RAL significantly increased BMD(P < 0.01),BV/TV(P <0.001),and Tb.N(P < 0.05)and decreased SMI(P < 0.001)and Tb.Sp(P <0.05)compared with those in the ACLT + OVX group.Part Three The study on the mechanism of raloxifene in the treatment of osteoarthritis induced by anterior cruciate ligament transection combined with ovariectomy in ratsObjective: This study was designed to investigate the mechanism of raloxifene(RAL)against osteoarthritis(OA)induced by a combination of ovitectomy(OVX)and anterior cruciate ligament transection(ACLT)in rats,so as to provide theoretical support for the clinical application of RAL in the treatment of postmenopausal OA.Methods:1.30 twelve-week-old female Sprague-Dawley rats were randomly divided into five groups as follows: The sham group,ACLT group,OVX group,ACLT + OVX group,and RAL group(ACLT + OVX +RAL)(n = 6).2.The model of osteoarthritis(OA)was induced by a combination of ovitectomy(OVX)and anterior cruciate ligament transection(ACLT).Rats in the OVX and ACLT groups were underwent bilateral OVX and ACLT on the right knee,respectively.The ACLT + OVX and RAL groups received both OVX and ACLT,and the sham group was underwent only sham operation.The rats in the RAL group received RAL by oral gavage(6.25 mg/kg)daily at72 hours after the surgery for 12 weeks.Other groups received the same volume of distilled water as a vehicle.3.After sacrifice,the right knees were fixed,decalcified,embedded,and sectioned,sequentially.The protein levels of type X collagen(Col-X),a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5),cysteinyl aspartate specific proteinase-3(caspase-3),matrix metalloproteinase-13(MMP-13),transforming growth factor-beta 1(TGF-?1)in the cartilage and the level of type I collagen(Col-I)in the subchondral bone were evaluated with integratal optical density(IOD)by immunohistochemistry.Furthermore,tartrate-resistant acid phosphatase(TRAP)staining was performed to evaluate the osteoclast activity in the subchondral bone.Results:1.Immunohistochemical results showed that the levels of MMP-13,ADAMTS-5,COL-X,caspase-3,and TGF-?1 in the ACLT group were markedly higher than those in the sham group(all P < 0.05)but lower than those in the ACLT + OVX group(all P < 0.05).However,the levels of these factors were substantially decreased after RAL treatment compared with those in the ACLT + OVX group(all P < 0.05).The expression levels of MMP-13 and TGF-?1 in the OVX group were higher compared with those in the sham group(all P < 0.05),but lower than those in the ACLT + OVX group(all P <0.05).The levels of other factors in the OVX group were higher than those in the sham group,but there were no significant differences(all P > 0.05).In addition,the level of Col-I in the OVX group was significantly lower than that in the sham group(P < 0.001)but considerably higher than that in the ACLT+ OVX group(P < 0.001).Conversely,RAL increased the level of Col-I compared with that in the ACLT + OVX group(P < 0.001).No marked difference was detected between the ACLT and sham groups(P > 0.05).2.The result of TRAP staining showed that no difference in the number of osteoclasts was detected between the ACLT and sham groups(P > 0.05).The number of osteoclasts was higher in the OVX group than that in the sham group(P < 0.05)and lower than that in the ACLT + OVX group(P < 0.001).RAL substantially decreased the number of osteoclasts compared with that in the ACLT + OVX group(P < 0.001).Conclusions:1.The model induced by ACLT combined with OVX showed severe degrees of the cartilage degeneration,subchondral bone resorption and microstructural damage,which shared similar pathological changes with human postmenopausal OA and could be used as a model for the studies on postmenopausal OA.2.Early administration of RAL retarded the destruction of the cartilage and subchondral bone thus effectively delaying the progression of OA induced by ACLT and OVX in rats,suggesting that RAL has the potential of clinical application in the prevention and treatment of postmenopausal OA.3.RAL may retard cartilage destruction of OA induced by ACLT and OVX in rats via restraining the overexpression of TGF-?1 and caspase-3,and improve the health of the subchondral bone by inhibiting osteoclast activity. |
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