The Study Of Sclerostin And HMGB1 In Osteoarthritic Cartilage And Subchondral Bone Degeneration

Objective:To study the expression and effect of HMGB1 in cartilage,Sclerostin in the subchondral bone in osteoarthritis.Methods:A total 20 cases of medial apartment dominated varus knee osteoarthritis were involved in this study.There were 8 males and 12 females with an average age of 67.8 years(range from 61 to 78 years).They had an average of 3.2 years of osteoarthritis(range from 2 to 5 years).Before operation,the knee joint X-ray films were measured,and the varus angle was 12.0 to 25.5 degrees,with an average of 17.6degrees.All these 20 cases were grade(5 cases)and grade(15 cases)osteoarthritis according to Kellgren–Lawrance classification.Tibial plateaus were obtained during total knee arthroplasty.A total 20 cases of type femoral neck fracture were involved in this study.The average age was 60.4 years old.Cartilage from the femoral head and the medial part of the plateaus were studied and the expression of HMGB1 was tested respectively.Took the medial and lateral subchondral bone Micro-CT scan,compared the changes in bone structure,calculated of Bone Volume/Total Volume(BV/TV),Trabecular Number(Tb.N),Trabecular Thickness(Tb.Th),Structure Model Index(SMI),and the Trabecular Separation(Tb.Sp).Immunohistochemistry and real-time PCR were used to test the expression of sclerostin and sost gene in subchondral bone of the tibial plateaus.Analysis the correlation between the expression level of sclerostin of subchondral bone and osteoarthritis K-L score,WOMAC score and bone structure parameters change.Analysis the correlation between the expression level of HMGB1 in cartilage and OARSI cartilage grade.The chondrogenic differentiation of BMSCs in the presence and absence of HMGB1 was detected.Briefly,BMSCs(1×10~5 cells/well)were plated onto 24-well plates and incubated at 37 until the confluence reached 80%.Then,the culture medium of BMSCs was replaced by chondrogenic-inductive medium in the presence or absence of HMGB1.The composition of chondrogenic-inductive medium was changed every two days.The induction of chondrogenic differentiation of BMSCs was persisted for 7 days,and then the resulted BMSCs were collected for safranin O staining and quantitative real-time PCR.Results:Compared with normal cartilage,the expression of HMGB1 in osteoarthritic cartilage was higher(p<0.05).Micro-CT found a significant rise of BV/TV,Tb.N and Tb.Th in the medial subchondral bone compared to the lateral part(p<0.05),but a reduction of SMI and Tb.Sp(p<0.05).Real time fluorescence quantitative PCR detection of sost gene,with the medial platform expression level for reference(2–??Ct:1.000),and the level of lateral platform expression(4.157±2.790)was significantly lower,the difference was statistically significant(t=2.263,P 0.001).Microscopic observation showed that the number of Sclerostin positive cells was significantly increased in the subchondral bone of the lateral tibial plateau.The percentage of Sclerostin positive cells in the lateral subchondral bone was 52%±0.19%,which was significantly higher than that of the medial platform subchondral bone(7.20%±0.04%),and the difference was statistically significant(t=5.094,P=0.005).The expression of sclerotic protein in subchondral bone was negatively correlated with K-L grade,WOMAC score and bone mass of osteoarthritis.The expression level of HMGB1 in cartilage was positively correlated with the grade of cartilage OARSI.BMSCs were stimulated with 0,0.5 or 1?g/ml recombination HMGB1,and the expressions of chondrogenic differentiation-associated genes were determined at 7 d after stimulation.The results showed mRNA levels of Sox9,type II collagen were all remarkably decreased by 0.5 or 1?g/ml recombination HMGB1compared with non-treated cells(P<0.05).In addition,the expression levels of ADAMTS4,ADAMTS5,ALP and type X collagen were all remarkably increased by stimulated with HMGB1.The promotive effect of differentiation culture medium on proteoglycan synthesis was largely reduced following HMGB1 treatment in BMSCs as indicated by Safranin O staining.The results was consistent with corresponding genes.Conclusion:Increasing expression of HMGB1 in cartilage,decreasing expression of Sclerostin in subchondral bone play an important role in osteoarthritis.HMGB1 could attenuates chondrogenic differentiation of BMSCs.The changes of these molecules expression levels may serve as a new target for intervention and treatment of osteoarthritis in the future.