| Objective The changes of articular cartilage and subchondral bone in different stages of degenerative knee osteoarthritis are systematically studied in order to confirming that intraosseous pressure is a key factor which is closely related to the pathological degenerative process of articular cartilage and subchondral bone.intraosseous pressure intervention can effectively curb the degeneration progress of articular cartilage and subchondral bone and is provided as a new surgical method for early treatment of knee osteoarthritis.Methods A series of months age(3,6,9,12,18)Dunkin-Hartley guinea pigs(N=10)were studied to get their body-weight-to-months-age curve,knee joint radiographic data including X-rays,Micro CT and MRI.The results of X-rays and MRI images were analyzed by Kellgren-Lawrence Osteoarthritis Score system and Osteoarthritis MRI Reference Image score system respectively and the reasonable month-age spectrum of Dunkin-Hartley guinea pigs for osteoarthritis reaserch was determined.In different month-age(6,9,12,18)groups of Dunkin-Hartley guinea pigs(N=5),intraosseous pressure in the distal femur and theproximal tibia were recorded and articular cartilage changes located in the weight bearing zone of the knee were observed in histology(gross,HE,toluidine blue)and immunohistochemistry(type 2 Collagen,MMP13).Intraosseous pressure were also recorded at the decompression moment and at 12 weeks after the intervention of bone pressure in different month-age(6,9,12,18)groups of Dunkin-Hartley guinea pigs(N=5)and different degenerative degree of articular cartilage and subchondral bone in the decompressed side and non-decompression side were compared using Micro CT scanning,histological method(gross,HE,toluidine blue)and immunohistochemistry technique(Type 2 collagen and MMP13).All data were analyzed by SPSS13.0 statistical software and Graph Pad Prism5.01 software.The differences of body weight in different months age(3,6,9,12,18)groups were statictically analyzed by one-way ANOVA.Kellgren-Lawrence Osteoarthritis score differences and Osteoarthritis MRI Reference Image score differences among those groups was statistically analyzed by one-way ANOVA and the t-test was used to compare the differences between groups.The differences of histological staining scores of articular cartilage in different months age(6,9,12,18)groups were analyzed by one-way ANOVA and between groups the t-test was used to compare the differences.The difference of histological staining scores between two sides of the knee in each group(6,9,12,18)was compared with the independent sample t test.One-way ANOVA was used to compare the intraosseous pressure changes of the proximal femur and the proximal tibia in different month-age groups(6,9,12,18)and the t-test was used to compare the differences between groups.In each month-age group(6,9,12,18),intraosseous pressure changes of the proximal femur and/or the proximal tibia were compared using independent samples t test.Above all,the test levels were set to both sides(? = 0.05).Results The body weight of Dunkin-Hartley guinea pigs increased gradually as month-age became older,and weight increasing was more rapidly before 9 months and was slowed down after 9 months of age.The differences of body weight between groups was statistically significant(F=433.892,P=0.000).The Kellgren-Lawrence knee Osteoarthritis scores increased gradually as month-age older,and the difference between groups was statistically significant(F=219.802,P=0.000).There was no significant difference in the scores between 3 months age and 6 months age(P = 0.068).There was significant difference between the other months age groups(P = 0.000).The scores of Osteoarthritis MRI reference Image were significantly increased as above(F = 303.840,P = 0.000).There was no significant difference in the scores between 3 months age and 6 months age(P=1.000)and was significant difference between the other months age groups(P=0.000).Micro CT showed that subchondral bone collaps and fusion in weight-bearing areas of distal femur and tibial proximal tibia from 9 months old to 18 months old Dunkin-Hartley guinea pigs.Mankin scores(HE,toluidine blue O)increased gradually with month age becoming older,and the difference between groups was statistically significant(P = 0.000),which were covering the different degenerative phases of knee osteoarthritis.The difference of both sides in each group had no statistically significant(P>0.05).Mankin scores(type 2 collagen,MMP13)increased gradually with with month age older,,and there was significant difference(P=0.000)between different month-age groups.Among them,there was significant difference between 6 months age and 12 months age(P<0.01)and difference between 12 month age and 18 months age(P<0.05).The score difference of both sides in each group had no statistically significant(P>0.05).The change trend of intraosseous pressure in distal femur and proximal tibia was increasing firstly and then climbed down with the increase of months age and there was significant statistical difference(femoral side: F=8.261,P=0.000;tibial side: F=8.469,P=0.000).Among them,intraosseous pressure was slightly increased in 9 month-age group than 6 month-age group,but there was no significant difference between them(femoral side: P=0.997;tibial side: P=0.973).At 12 months age,intraosseous pressure was significantly higher than that of 9 month-age group(femoral side: P=0.019;tibial side: P=0.019).When to 18 months age,intraosseous pressure became lower than the 12-month-old and the difference was statistically significant(femoral side: P=0.000;tibial side: P=0.000).In terms of each group,there was no significant intraosseous pressure difference between distal femurs or proximal tibias(P>0.05).The intraosseous pressure of distal femur was slighrly higher than that of proximal tibia,but there was no significant difference between them(P>0.05).At 12 weeks postoperatively,whether decompression or not in different month-age(6,9,12,18)groups,intraosseous pressure of distal femur and proximal tibia were also presented climing up firstly and then climbed down with months age increasing,but there was no statistical significance among group differences(decompression side: femoral F=0.623,P=0.611,tibial F=2.230,P=0.124;non-decompression side: femur F=0.690,P=0.571,tibia F=2.214,P=0.126).Intraosseous pressure of decompression side were lower than that of non-decompression side,but the difference had no statistically significant(P>0.05).Intraosseous pressure of distal femue was lower than that of proximal tibia in the decompression side but the difference was not statistically significant(P>0.05).In non-decompression side,the distal femoral intraosseous pressure was also lower than that of the proximal tibia,although the difference was not statistically significant(P>0.05).The distal femoral intraosseous pressure was lower obviously compared with that of proximal tibia at immediate decompression in different month-age(6,9,12,18)groups.Postoperative immediate intraosseous pressure difference itself and decling range compared with the preoperative intraosseous pressure,all had significant difference(P = 0.000).At 12 weeks after decompression surgery,histological findings showed that both knee joints of Dunkin-Hartley guinea pigs in different month-age groups(6,9,12,18)had articular cartilage degeneration.However,the degenerative extent were significantly reduced in decompression side than in non-decompression side.The Mankin scores(HE,toluidine blue O)also gradually increased with months age,but the degeneration degree and level in all decompression sides was significantly climbing down and reduced.In each month-age group,the differences were statistically significant between decompression side and non-decompression side(P<0.01).Mankin scores(type 2 collagen)gradually increased with the increase of months age.The positive expression thickness,content and hierarchical distribution characteristics of type 2 collagen were effectively retained in decompression sides.There was significant statistical significance between decompressed and non-decompressed sides in each month-age group and the stress reduction side of the stress side(6DH: t=-3.500,P=0.008;9DH: t=-3.497,P=0.008;12DH: t=-6.668,P=0.000;18DH: t=-6.396,P =0.000).Mankin scores(MMP13)gradually ascended with the increase of months age and the decompression sides were effectively reduced the expression of MMP13 in articular cartilage than the non-decompression side.The expression of MMP13 in articular cartilage was significantly lower in decompression side than that in the control group(6DH: t=-3.795,P=0.005;9DH: t=-4.427,P=0.002;12DH: t=-3.883,P = 0.005;18DH: t =-4.333,P = 0.003).Micro CT showed that in non-decompression sides,subchondral trabecular bone arrangement were more sparse than that in decompression sides,subchondral cysts in distal femur and proximal tibia were more obvious,more extensive range of erosion in non-decompression sides.Subchondral cysts can be located in the distal femur and tibial proximal intercondylar sites,medial condyles and lateral condylars where weight-bearing areas were located.If subchondral cysts expanded and invade to subchondral bone plate,there may be happened subchondral bone plate fractures.Conclusion As a sequence of primary degenerative knee osteoarthris animal model,Dunkin-Hartley guinea pigs in different month-age(6,9,12,18)groups had shown little differences in each group and a gradient changes among groups.Thus it is reliable applying the month-age(6,9,12,18)series of Dunkin-Hartley guinea pig in studying different pathological stages of knee osteoarthris.Characteristical changes of intraosseous pressure in distal femur and proximal tibia met well with degenerative progress of knee osteoarthris,which had been revealing that there might be a corerelation betwen the changes of intraosseous pressure and the pathological changes of articular cartilage and subchondral bone.The intervention surgery to intraosseous pressure could effectively protect the morphological structure of articular cartilage and subchondral bone in different knee osteoarthris stages of Dunkin-Hartley guinea pigs,reduce the destruction of type 2 collagen and decrease the expression of MMP13 in articular cartilage.The minimally invasive,targeted,standardized surgical technique of intraosseous pressure intervention has a good clinical application prospects. |
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