| Part one The experimental study of lumbar facet joint degeneration induced by intra-articular injection of type II collagenase in ratsObjective: This study was aimed to establish an animal model of facet joint osteoarthritis(FJ OA)induced by intra-articular injection of type II collagenase in rats,and to evaluate the degenerative changes of articular cartilage and subchondral bone using histological method and micro-CT analysis.Methods: Twenty 3-month-old male Sprague-Dawley(SD)were randomly assigned to the Sham group and the CI group(n=10 for each group).Rats in the Sham group received sham surgury,and rats in the CI group received intra-articular FJ injections of type II collagenase.After 8 weeks of surgery,all animals were euthanized.The evaluation of histological changes of FJ cartilage and scoring were performed using toluidine blue staining.The histological changes of subchondral bone were evaluated by HE staining.The microarchitecture parameters of subchondral bone were analyzed with micro-CT scan.Results: The results of toluidine blue staining showed that the articular cartilage surface was smooth and regular,the morphology of chondrocyte was normal,and the matrix of cartilage was uniformly stained with toluidine blue in the Sham group.The articular cartilage in the CI group displayed obvious degeneration,characterized by fissures extending into the middle zone of cartilage and reduced numbers of chondrocytes.The histological score in the CI group was significantly higher than that in the Sham group(P < 0.001).The HE staining showed that the structure of subchondral bone was normal and the arrangement of subchondral trabeculae was orderly in the Sham group,whereas the CI group displayed obvious lesions in the subchondral bone with focal fibrous tissue proliferation(cyst).The micro-CT analysis displayed the right superior articular process in the Sham group possessed a normal structure and developed a slender shape,whereas the CI group showed a hypertrophic appearance.The CI group had significantly lower BMD,BV/TV,and Tb.Th values than the Sham group(all P < 0.001),whereas the Tb.Sp and SMI values were significantly higher(P = 0.003 and P< 0.001).Statistically significant differences in the Tb.N were not observed between groups(P = 0.177).Part two The study of the effects of salmon calcitonin on lumbar facet joint articular cartilage degeneration induced by intra-articular injection of type II collagenase in ratsObjective: This study was aimed to use salmon calcitonin(s CT)and celecoxib(CLX)to treat lumbar facet joint osteoarthritis(FJ OA)induced by intra-articular injection of type II collagenase in rats,and to evaluate the effects of these two drugs on articular cartilage degeneration of facet joint.Methods: Forty 3-month-old male Sprague-Dawley(SD)were randomly assigned to the Sham group,the CI group,the CLX group and the sCT group(n=10 for each group).Rats in the Sham group received sham surgury,and rats in the CI group,the CLX group and the sCT group received intra-articular FJ injections of type II collagenase.After surgery,rats in the CI group received a subcutaneous injection of normal saline,rats in the sCT group received a subcutaneous injection of sCT at a dose of 16 IU/kg/2 days,and rats in the CLX group received oral gavage of CLX at a dosage of 50 mg/kg/day.After 8 weeks of treatment,all animals were euthanized.The evaluation of degenerative changes of FJ cartilage and scoring were performed using toluidine blue staining.The expression levels of aggrecan(AGG),collagen-II(Col-II),caspase-3,metalloproteinase-13(MMP-13),and a disintegrin and metalloproteinase with thrombospondin motifs 4(ADAMTS-4)in FJ articular cartilage were investigated by immunohistochemical methods.Serum concentrations of cartilage oligomeric matrix protein(COMP)and MMP-13 were determined using enzyme-linked immunosorbent assay(ELISA)kits.The correlation between modified Mankin score and serum COMP was investigated by the methods of correlation analysis.Results: The results of toluidine blue staining showed that the articular cartilage surface was smooth and regular,the morphology of chondrocyte was normal,and the matrix of cartilage was uniformly stained with toluidine blue in the Sham group.The articular cartilage in the CI group displayed significant degeneration,characterized by reduced numbers of chondrocytes,fissures extending into the middle zone of cartilage,and even denudation of the articular surface.After treatment of CLX,the degeneration of cartilage in the CLX group was alleviated to some extent,but there were still obvious surface lesions and local loss of staining in cartilage.In the sCT group,the surface fissures of cartilage were significantly reduced,the number of chondrocyte was normal,the extracellular matrix was uniformly stained with toluidine blue,and the degeneration of cartilage was significantly attenuated.The CI group displayed a significantly higher histological score than the Sham group(P < 0.001),and the s CT group exhibited a significantly lower histological score than the CI group(P = 0.034).Caspase-3 was expressed at significantly higher levels in the CI group than in the Sham group(P < 0.001).Lower caspase-3 expression was observed in the sCT and CLX groups than in the CI group(P < 0.001 and P = 0.010,respectively),whereas the sCT group expressed lower levels of this protein than the CLX group(P < 0.001).Significantly lower AGG expression was observed in the CI group than in the Sham group(P < 0.001).The s CT group showed a significant increase in AGG expression compared to that in the CI and CLX groups(P < 0.001 and P = 0.021,respectively),whereas no difference was observed between the CI and CLX groups(P = 0.097).ADAMTS-4 was expressed at significantly higher levels in the CI group than in the Sham group(P < 0.001),but ADAMTS-4 was expressed at significantly lower levels in the sCT group than in the CI and CLX groups(P < 0.001 and P = 0.001,respectively).No significant differences at the levels of ADAMTS-4 were observed between the CI and CLX groups(P = 0.103).Levels of Col-II and MMP-13 were not significantly different among the groups(P = 0.489 and P = 0.753,respectively).The serum COMP concentration was significantly higher in the CI group than in the Sham and sCT groups(P = 0.010 and P = 0.031,respectively),whereas no differences were observed between the CLX and CI groups(P = 0.282).The serum MMP-13 concentration was not significantly different among groups(P = 0.055).Significant positive correlation was observed between the serum COMP concentration and the modified Mankin score(r = 0.444,P = 0.004).Part three The study of the effects of salmon calcitonin on lumbar facet joint subchondral bone degeneration and related pain induced by intra-articular injection of type II collagenase in ratsObjective: This study was aimed to use salmon calcitonin(s CT)and celecoxib(CLX)to treat lumbar facet joint osteoarthritis(FJ OA)induced by intra-articular injection of type II collagenase in rats,and to evaluate the effects of these two drugs on subchondral bone degeneration of FJ and related pain.Methods: Forty 3-month-old male Sprague-Dawley(SD)were randomly assigned to the Sham group,the CI group,the CLX group and the sCT group(n=10 for each group).Rats in the Sham group received sham surgury,and Rats in the CI group,the CLX group and the sCT group received intra-articular FJ injections of type II collagenase.After surgery,rats in the CI group received a subcutaneous injection of normal saline,rats in the sCT group received a subcutaneous injection of sCT at a dose of 16 IU/kg/2 days,and rats in the CLX group received oral gavage of CLX at a dosage of 50 mg/kg/day.After 8 weeks of treatment,all animals were euthanized.The tactile allodynia test was performed before surgery(days 0)and on days 3,7,14,28,42 and 56 after surgery using a calibrated set of von Frey filaments.After all animals were euthanized,the microarchitecture parameters of subchondral bone were analyzed with micro-CT scan.The micro-CT image of the L4–L5 FJ was used to measure the FJ space as well as the length(L),height(H)and L/H ratio of the superior articular process,and to conduct micro-CT scoring.The correlations between micro-CT score and modified Mankin score,and between serum COMP and micro-CT score were investigated by the methods of correlation analysis.Results: The tactile allodynia was not observed in sham-operated rats throughout the experiments.The other three groups displayed no tactile allodynia before surgery(days 0).On days 3 after surgery,these three groups showed severe tactile allodynia;the CLX group displayed a significantly higher 50%PWT value than the CI group(P <0.001),but there were no significant difference between the CLX group and the sCT group(P =0.146)and between the sCT group and the CI group(P =0.122).On days 7 after surgery,the 50%PWT value of the CLX group or the sCT group was significantly higher than that of the CI group(P = 0.004 and P = 0.041,respectively),but there were no significant difference between the CLX group and the s CT group(P =1.000).On days 14 after surgery,the 50%PWT value of the CLX group or the sCT group was significantly higher than that of the CI group(P = 0.021 and P = 0.002,respectively),no significant defference were found between the CLX group and the sCT group(P =1.000).On days 28 after surgery,the 50%PWT value of the sCT group was significantly higher than that of the CI group(P = 0.002),but statistical differences were not observed between the CLX group and either the CI or sCT group(P = 0.295 and P = 0.213,respectively).On days 42 and 56 after surgery,the s CT group displayed significantly higher 50% PWT values than that of the the CI group and the CLX group(P = 0.043 and P = 0.025;P < 0.001 and P = 0.020,respectively),but statistical differences were not observed between the CLX group and the CI group(P = 1.000 and P = 0.625,respectively).The BMD,BV/TV,and Tb.Th values were significantly decreased in the CI group compared to those in the Sham group(all P < 0.001),but the Tb.Sp and SMI were significantly increased(P = 0.002 and P = 0.013,respectively).The sCT group displayed significantly higher BMD,BV/TV,and Tb.Th values than the CI group(P = 0.003,P = 0.001,and P = 0.040,respectively),whereas the Tb.Sp and SMI were significantly lower(P = 0.019 and P = 0.031,respectively).The sCT group exhibited significantly higher BMD and BV/TV values than the CLX group(P = 0.016,and P = 0.004,respectively),but statistical differences were not observed in the Tb.Th,Tb.Sp and SMI values between the CLX group and the sCT group(P = 0.175,P = 0.074 and P = 0.054,respectively).There were no significant differences in the BMD?BV/TV?Tb.Th?Tb.Sp and SMI values between the CI group and the CLX group(P = 0.541,P = 0.515,P = 0.459,P = 0.545 and P = 0.803,respectively).Statistically significant differences in the Tb.N were not observed between groups(P = 0.096).The right superior articular process in the Sham group developed a slender shape with a length that was significantly larger than its height,whereas the CI group showed a hypertrophic appearance,with a significantly increased height.Specifically,the CI group displayed a 13.81% decrease in length and a 58.87% decrease in the L/H ratio(all P < 0.001),but a 113.38% increase in height compared to the Sham group(P < 0.001).Compared to the CI and CLX groups,the sCT group exhibited 60.42% and 23.63% increases in the L/H ratio(P < 0.001,and P = 0.005,respectively),but 34.14% and 15.07% decreases in height,respectively(P = 0.001,and P = 0.098,respectively).The CLX group displayed a 29.76% increase in the L/H ratio but a 22.45% decrease in height compared with the CI group(P = 0.006,and P = 0.023,respectively).No significant differences in the joint space width were observed between groups(P = 0.376).Significantly higher micro-CT scores were recorded for the CI group than for the Sham and sCT groups(P < 0.001 and P = 0.002,respectively),but statistical differences were not observed between the CLX group and either the sCT or CI group(P = 0.685 and P = 0.230,respectively).Significant positive correlations were observed between the micro-CT score and the modified Mankin score(r = 0.807,P < 0.001),and between the serum COMP concentration and the micro-CT score(r = 0.370,P = 0.019).Part four The study of the effects of salmon calcitonin on articular cartilage degeneration of lumbar facet joint in ovariectomized ratsObjective: This study was aimed to explore the characteristics and mechanisms of FJ cartilage degeneration caused by estrogen deficiency,and the effects of salmon calcitonin(s CT)on FJ cartilage degeneration.Methods: Thirty 3-month-old female Sprague-Dawley(SD)were randomly assigned to the Sham group,the OVX group and the OVX+sCT group(n=10 for each group).Rats in the Sham group received sham surgury,and rats in the OVX group and the OVX+sCT group received bilateral ovariectomy.After surgery,rats in the OVX group received a subcutaneous injection of normal saline,rats in the sCT group received a subcutaneous injection of sCT at a dose of 16 IU/kg/2 days.After 12 weeks of treatment,all animals were euthanized.The evaluation of degenerative changes of FJ cartilage and scoring were performed using toluidine blue staining.The expression levels of collagen-II(Col-II),caspase-3,and metalloproteinase-13(MMP-13)in FJ articular cartilage were investigated by immunohistochemical methods.Serum concentrations of cartilage oligomeric matrix protein(COMP)were determined using ELISA kits.The correlation between modified Mankin score and serum COMP,and between cartilage thickness and serum COMP were investigated by the methods of correlation analysis.Results: A significantly higher serum COMP concentration was observed in the OVX group than in the Sham group(P < 0.001),whereas after s CT treatment,rats in the OVX+sCT group showed a significantly lower serum COMP concentration than that observed in the OVX group(P = 0.028).No significant difference in serum COMP was observed between the Sham group and the OVX+sCT group(P > 0.05).The results of toluidine blue staining showed that the structures of chondrocytes were normal,and toluidine blue staining of extracellular matrix(ECM)was evenly distributed.Distinct cartilage degeneration was observed in the OVX group,characterized by surface irregularities and small fissures.After treatment with sCT,cartilage lesions of OVX rats were alleviated to some extent.The OVX group showed a significantly higher modified Mankin score than did the Sham group(P = 0.015).No significant differences were found between the OVX group and the OVX+sCT group or between the Sham group and the OVX+s CT group(all P > 0.05).The cartilage thickness of the OVX group decreased significantly compared with that of the Sham group(P < 0.001).The OVX+sCT group presented a significantly greater cartilage thickness than that of the OVX group(P = 0.024),but displayed a significantly less cartilage thickness than that of the Sham group(P = 0.007).Significantly higher MMP-13 and caspase-3 expression levels were observed in the OVX group than in the Sham group(all P < 0.001),whereas significantly lower levels of MMP-13 and caspase-3 expression were observed in the OVX+sCT group than in the OVX group(all P < 0.001).The OVX+sCT group showed no significant differences in the expression levels of MMP-13 and caspase-3 compared with the Sham group(all P > 0.05).Levels of Col-II were not significantly different among the groups(P > 0.05).The serum COMP concentration showed a significant positive correlation with the modified Mankin score(r = 0.467,P < 0.05)but a significant negative correlation with cartilage thickness(r =-0.485,P < 0.05).Conclusions:1.Intra-articular FJ injections of type II collagenase could cause hypertrophic changes of articular process,obvious lesions in articular cartilage,and fibrous tissue proliferation(cyst)and microarchitectural degeneration in subchondral bone.2.Both CLX and sCT have protective effects on cartilage of FJ OA induced by intra-articular injections of type II collagenase,but sCT has more significant protective effects,which may be attributed to the ability of sCT in maintaining metabolic balance of cartilage and inhibiting apoptosis of chondrocytes.Besides,serum COMP may possess the potential in predicting FJ OA.3.Intra-articular FJ injections of type II collagenase could cause obvious tactile allodynia,hypertrophy of articular processes,increased micro-CT score,and subchondral bone degeneration.Both sCT and CLX have some analgesic effects,but s CT possesses better long-term analgesic effects.Moreover,s CT could significantly decrease micro-CT score,inhibit subchondral bone degeneration and hypertrophy of articular processes.4.Estrogen deficiency could lead to obvious FJ cartilage degeneration 12 weeks after ovariectomy,especially significantly decreased cartilage thickness.sCT could inhibit enhanced catabolic activities of cartilage and increased serum concentrations of COMP,and has protective effects on FJ cartilage. |
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