| Objective:(1)From the perspective of TGF-β1,to explore the possible clinical significance of serum markers of normal and early osteoarthritis patients and bone marrow lesion in MRI.To analyze the expression of TGF-β1 in the intact and partial defects of articular cartilage of the tibial plateau in patients with osteoarthritis and the possible role of TGF-β1 in osteoarthritis.(2)Through the establishment of a canine model of experimental osteoarthritis,to expound the early diagnosis value of serum biomarkers and MRI,and explore the mechanism of the role of TGF-β1 in OA.(3)By establishing the canine model of experimental osteoarthritis,local subchondral bone injection of halofuginone,to explore the effect of halofuginone on OA biology and imaging and its mechanism.Methods:(1)By comparing the serum IIA procollagen amino-terminal peptide(PIIANP)and type X collagen α1 chain(COL10A1),which is the metabolite of type II collagen and type X collagen,between normal and early osteoarthritis patients,and in combination with the relationship between the pain symptoms of patients with early osteoarthritis and bone marrow lesion BML in MRI,to verify the early serum and imaging changes in OA.To select two kinds of specimen(Patient’s tibial plateau in knee arthroplasty),where articular cartilage is either intact or partial degenerated,by observing the morphology of the tibial plate.Safranin OFast Green staining,OARSI-modified manking score and immunohistochemistry were used to detect the expression of MMP-13 and TGF-β1 in articular cartilage.Micro-CT was used to compare the microstructure of subchondral bone in two specimens,Verify the status of articular cartilage in two specimens.(2)To establish osteoarthritis model(OA was surgically induced via anterior cruciate ligament transection(ACLT)+ medial meniscectomy)group and Sham control group by establishing an animal model of adult beagle dogs,and Longitudinal studies at 4 weeks,8 weeks,12 weeks and 16 weeks after surgery were performed to measure the levels and changes of serum CTX-II and COL10A1 serum markers,and the tibial plateau cartilage volume was quantitatively assessed by MRI software to measure changes in the volume of articular cartilage between the two groups over time.The beagle dogs were sacrificed 16 weeks after operation,and the structures of the cartilage and subchondral bone were observed by micro-CT scanning,and the changes of the cartilage and subchondral bone microstructure in OA model were observed.Based on Micro-CT tests,combined with the Safranin O-Fast Green staining,OARSI-Modified Manking score were used to evaluate the degree of articular cartilage damage.The expression of TGF-β1 in articular cartilage of the two groups was observed by immunohistochemistry.(3)To establish osteoarthritis model group,Sham control group and the halofuginone treatment group by establishing an animal model of ACLT + medial meniscectomy in adult beagle dogs.Longitudinal studies at 4 weeks,8 weeks,12 weeks,and 16 weeks after surgery were performed to measure the level and change of serum CTXII and COL10A1 serum markers.The volume of tibial plateau cartilage was quantitatively evaluated by MRI software,and the changes of articular cartilage volume between the treatment group and the other two groups were measured over time.The beagle dogs were sacrificed 16 weeks after operation,and the structures of the cartilage and subchondral bone were observed by Micro-CT scanning,and the changes of the cartilage and subchondral bone micro structure in OA model were observed.Based on Micro-CT tests,combined with the Safranin O-Fast Green staining,OARSI-Modified Manking score and hematoxylin eosin staining were used to evaluate the degree of articular cartilage damage.The expression of TGF-β1 in articular cartilage of the two groups was observed by immunohistochemistry.Results:(1)The serum concentration of COL10A1 in knee OA group(1.30±0.65)ng / ml was significantly higher than that in control group(0.46±0.15)ng / ml,and the difference was statistically significant(P<0.01).The level of serum PIIANP in knee osteoarthritis group(13.22 ± 4.74)ng / ml was lower than that in control group(23.88 ± 4.68)ng / ml,and the difference was statistically significant(P<0.01).The serum level of COL10A1 in the control group was lower than that in the knee osteoarthritis group,and the difference was statistically significant(P<0.05).The serum PIIANP level in the control group was higher than that in the knee OA subgroups,and the difference was statistically significant(P<0.01).There was a difference in the degree of bone marrow edema between the no-pain group and the pain group.The higher the grade of pain,the more severe the edema of the bone marrow(p<0.001).The comparison of any two samples among the grading of bone marrow lesion in the three different grades of pain indicates: The difference was statistically significant(p<0.01)in no pain group compared with mild pain group and moderate to severe pain group.Safranin O-Fast Green staining and OARSI-Modified Manking score suggested that the degree of cartilage destruction in the cartilage incomplete tibial plateau specimens was more severe than that in the intact specimens(p<0.01).Immunohistochemistry showed that the expression of MMP-13,TGF-β 1 and p-Smad 2/3in articular cartilage of articular cartilage partially abraded specimens was more.Micro-CT showed thatthe volume fraction of subchondral bone,the thickness of subchondral bone plate were higher and the trabecular pattern factor was lower in the cartilage partially abraded specimens(p<0.01).(2)Serum markers showed that COL10A1 levels peaked at W8,after which the level decreased,but remained significantly higher than the control group.Significant differences were detected between the OA and control groups at W8 and W12(all P<0.05).The highest level of the CTX-II marker was at W12,after which the levels decreased but remained significantly higher than the control group(P<0.05).Significant differences were observed between the OA and control groups at three of the time points,with the exception of W4 post-surgery(P=0.335,<0.01,<0.01,and<0.01 respectively).Quantitative MRI evaluation results showed that Compared with the control group at W8 after surgery,we found that the cartilage volume markedly decreased in the tibial plateau.Furthermore,the cartilage volume of the tibial plateau was significantly reduced at W16 post-surgery.Significant differences were found between the OA and control groups at W8 and W16 in Fig 5(all P-values<0.01).Significant differences were observed between the OA and control groups at different time intervals,with the exception of baseline and W4 values(P=0.659,0.436,<0.01 and<0.01,respectively).Comparing the postoperative score of Beagle dogs,the Lameness score of OA group was higher than sham group at W4,W8,W12 and W16 after operation,and the difference was statistically significant(P<0.01).Safranin O-Fast Green staining and OARSIModified Manking score suggested that compared with the sham group,the degree of cartilage destruction was even more serious in the model group,and the difference between the two groups was statistically significant(P<0.01).Immunohistochemistry showed that the expression of MMP-13 and TGF-β1 in articular cartilage of OA model group was higher.Micro-CT showed that the volume fraction of subchondral bone,the thickness of subchondral bone plate were higher,and the trabecular bone model factor was lower(p<0.01).(3)The level of COL10A1 in OA group was significantly higher than that in the treatment group and the sham group at W8 and W12,and the difference was statistically significant(P<0.05).There was no significant difference between the treatment group and sham group at W8 and W12(P=0.05).Significant differences of the level of the CTX-II marker were observed between the OA and control groups at three of the time points,with the exception of W4 post-surgery(P=0.335,<0.01,<0.01,and<0.01 respectively).The difference between OA group and sham group is statistically significant(P<0.05).There was no significant difference between the sham group and the treatment group(P>0.05),indicating that halofuginone delayed and prevented the hypertrophy of articular chondrocytes in the early stage of OA.By quantitative MRI examination,no clear bone marrow edema was found in the treatment group and the sham group,and 3 cases of bone marrow edema were found in the OA group.The cartilage volume of tibial plateau in OA group decreased significantly at W12 and W16 after operation,the difference between group OA and treatment group and group sham at W12 and W16 was statistically significant(all P<0.01).There was no significant difference between treatment group and sham group at W12 and W16,indicating that halofuginone played a role in protecting articular cartilage.Safranin O-Fast Green staining and OARSI-Modified Manking score suggested that the OARSI-Modified Manking score was higher in the OA model group than in the treatment group and sham group,indicating that the degree of cartilage destruction was more serious.and the difference is statistically significant compared with the other two groups(p<0.01).There was no significant difference between the treatment group and sham group(p>0.05).Immunohistochemistry showed that the expression of MMP-13 and TGF-β1 in the OA model group was higher than that in the treatment group and sham group,and the difference was statistically significant(p<0.01),and No differences were found betweenthe treatment group and the sham group.This also proves that the abnormal expression of transforming growth factor-β1 in the early stage of osteoarthrosis may be related to the occurrence of osteoarthrosis,and the articular cartilage was protected after local subchondral injection of halofuginone.Micro-CT examination showed an increase in subchondral bone volume fraction(BV/TV)in the OA group compared to the other two groups,the thickness of the subchondral bone plate(SBP.Th)increased,but trabecular pattern factor(Tb.Pf)decreased,the difference between OA group and the other two groups was statistically significant(p<0.01),and in the result of the above examination.No significant statistical difference was found between the treatment group and the sham group,indicating that the local subchondral bone injection of halofuginone played a protective role of subchondral bone.Conclusions:(1)The combined detection of COL10A1 and PIIANP expression with imaging KL classification system can predict the diagnosis and prognosis of early OA;the pain of knee osteoarthritis is related to the imaging change of MRI bone marrow edema and is of moderate correlation.,the higher the level of pain,the higher the level of bone marrow edema.In comparison with intact specimens of articular cartilage,the subchondral bone of degenerative,worn articular cartilage remodels abnormally and its microstructure changes,Meanwhile,the activity of TGF-β1 in subchondral bone also increases,which may become a target for the treatment of osteoarthritis.(2)In beagle dogs OA group and control group,detection of serum marker changes combined with quantitative MRI assessment of tibial plateau cartilage volume for monitoring the progress of OA,proves that the arthritis model of Beagle dog can be established successfully by amputation of anterior cruciate ligament transection.In the OA model group,the expression of transforming growth factor-β1 in articular cartilage was more,and the change of microstructure of subchondral bone resulted in the occurrence of osteoarthritis.(3)The serological biomarkers COL10A1 and CTX-II levels in beagle dogs OA group were significantly higher than that in the control group.The loss of articular cartilage was also found by quantitative measurement of cartilage by MRI.The abnormal increase of TGF-β 1 activity in articular cartilage was inhibited by local subchondral bone halofuginone injection,which delayed the progress of arthritis,corrected bone resorption and bone formation coupling.The abnormal bone resorption in the subchondral bone was inhibited,thus maintaining the stability of the microstructure of the subchondral bone and protecting the articular cartilage. |
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