| Background Anterior cruciate ligament(ACL)injury is an extremely common joint injury in the young population,and it places them at high risk for post-traumatic osteoarthritis(PTOA).Several new studies appear that the current gold standard treatment,surgical ACL reconstruction,does not appreciably reduce this risk.Since elevated levels of catabolic enzymes in synovial fluid induce chondrocytes death and cartilage matrix degeneration within one week of injury,early intervention strategies should focus on reducing these cartilage degrading enzymes within a similar time frame.Alpha-2-macroglobulin(A2M)is an endogenous inhibitor for majority cartilage catabolic cytokines(IL-1??6?8,TNF-a and GM-CSF)and cartilage degrading enzymes(MMP-3?9?13)in vitro and in vivo.Our recent findings showed that A2 M presented in early stage following anterior cruciate ligament-transected(ACLT)injury in synovial fluid,which was associated with PTOA,and A2 M as a master inhibitor of cartilage degrading factor can attenuate the progression of PTOA.However,the change of the concentration of A2 M in SF after ACL injury is unclear.Furthermore,it is not clear whether the multiple intra-articula injection can cause a serious side effect due to A2 M as foreign protein.Thus,developing a new method to purification or concentration of autologous A2 M from serum will be very vital for this application.Objectives 1)In this proposal study,we will verify A2 M as a potential early diagnosis biomarker of PTOA by measuring the time course of A2 M release in synovial fluid and establish its relationship with enzyme concentrations and cartilage damage after ACLT in the SD rat PTOA model.2)To analyze the molecular biology changes of A2M-Rich Serum(A2MRS)abtained by using ultrafiltration centrifugation methos.3)we will test whether supplemental intra-articular A2M-Rich Serum(A2MRS)injections will attenuate PTOA pathogenesis after ACLT in the SD rat modelMaterial and methods 1)Ninety-six male SD rats were divided into two groups randomly(n=48 per group).The animal model of PTOA was induced by anterior cruciate ligament transection(ACLT),as the experiment group and the same process of operation was performed without ACL cut as the sham control group.Eight rats in every group were sacrificed in different time points after operation,and A2 M concentrations in serum,synovial fluid(SF)were evaluated using enzyme-linked immunosorbent assay(ELISA)and cartilage degeneration were evaluated using safranin O staining.2)The molecular biology changes of A2M-Rich Serum(A2MRS)abtained by ultrafiltration centrifugation were performed,including protein activity and its concentration.3)In vivo effects on cartilage degeneration and matrix metalloproteinase 13(MMP-13)concentration were evaluated in male rats(n=80)randomized to 1 of 4 treatments:(1)ACLT+Saline;(2)ACLT+HA2MRS;(3)ACLT+LA2MRS;(4)Sham +Saline.Rats were administered intraarticular injections at day 3 after ACLT,and then every 2 weeks for 8 weeks.A mix of fluorescent imaging probes MMPSense680(10 ?l,13.3 ?M)and PBS(20?l)were injected intra-articularly 24 hours after A2 MRS injection,Fluorescence Molecular Tomography(FMT)was used to monitor the levels of inflammation(MMP-3,-9,and-13)in vivo 24 h after injection MMPSense 680.Gross morphologic lesions on the tibial plateau in rats were visualized by India ink staining 8 weeks after operation.The gross morphologic of OA progression was graded by Mankin's score.Safranin O,hematoxylin-eosin(HE)and toluidine blue staining were adopted to evaluate the degeneration of cartilage.Cartilage histologe degradation was quantified using the Osteoarthritis Research Society International(OARSI)grading system.OA-related gene expression was quantified by real-time quantitativepolymerase chain reaction.The concentration of MMP-13 in SF lavage fluid was measured using ELISA.Results 1)There were obviously cartilage degeneration of ACLT group compared with sham group after 8 weeks.The concentration of A2 M in synovial fluid(SF)was higher compared with in serum in ACLT group at 3 days,1 week and 8 weeks after operation.The concentration of A2 M in synovial fluid(SF)of ACLT group was higher compared with sham group at 3 days after operation,and rapidly decline after 1 week.2)ELISA results showed that the concentration of A2 M in A2 MRS was 19.52±4.366(mg/ml)as compared to 2.432±6.648(mg/ml)in normal serum and 1.416±4.934(mg/ml)in filtration fluid.SDS-PAGE gel stained with Coomassie blue showed a more prominent band(180 kd)in A2M-Rich Serum(A2MRS)than in normal serum and filtration fluid.The increase in A2 M levels was validated in A2 MRS as compared to normal serum and filtration fluid using Western blotting.Protein activity of A2 M in A2 MRS had increased 2.13 multiple compared with A2 M of normal serum.3)Matrix metalloproteinase(MMP-3?13)in SF of PTOA were obviously inhibited by A2 MRS,as detected by Fluorescence Molecular Tomography in vivo.Radiography indicated an obvious formation of the osteophyte at the patella in ACLT+Saline group.However,no obvious degenrations were observed in others groups.Decreased India ink staining and a smooth surface with stronger Safranin O staining were detected in the articular cartilage of A2MRS-treated animals as compared to untreated controls.There were not obvious damage of cartilage in Sham+Saline treated animals.Mankin's score showed that there was a significant difference between ACLT+Saline group and ACLT+A2MRS group,while there was no significant difference between ACLT+HA2MRS group and ACLT+LA2MRS group.After treatment with A2 MRS in ACLT,stronger Safranin O staining,more cellularity but less chondrocyte cloning,and less fibrillation were observed than that in the saline-treated groups.Cartilage from rats administered the HA2 MRS injection had stronger staining and more intact surface than the cartilage from rats administered the Low A2 MRS injection,but weaker staining than that from control rats that underwent sham operation.The same results were observed in hematoxylin-eosin(HE)and toluidine blue staining.OARSI histologic grading system scores in both A2MRS-treated groups suggested mild degeneration(Mean ±SD 5.54±2.61 in ACLT+HA2MRS group and 4.87±2.74 in ACLT+LA2MRS,P =0.22),while cartilage damage in rats that underwent ACLT and saline treatment was significantly more severe(Mean ±SD 10.15±2.88;P <0.001).The cartilage from rats that underwent sham operation had the least damage(Mean ±SD 3.05±1.39;P <0.001).Type II collagen expression in articular cartilage was higher in the A2M-treated and the sham-operated rats than that in rats that underwent ACLT and saline treatment.In contrast,matrix metalloproteinase 13(MMP-13)and type X collagen staining were elevated in rats that underwent ACLT and saline treatment,but was lower in the A2M-treated and sham-operatedrats,which is consistent with reduced OA damage in these rats.Real-time q PCR results indicated that supplemental intraarticular A2 MRS enhanced the levels of m RNA for Col II and Aggrecan,and suppressed the levels of m RNA for MMP 3,MMP 13,Runx2,and Col X in the rat ACLT model.Col II m RNA levels and Aggrecan levels in rats that underwent ACLT and saline treatment were lower than those in rats that underwent ACLT and were administered A2 MRS and those in rats that underwent sham operation,however there were no significant difference among 4groups in the two targets.In contrast,levels of m RNA for MMP 3,MMP 13,Runx2,and Col X in rats that underwent ACLT and saline treatment had the highest level among the 4 groups.In rats that underwent ACLT and saline treatment,the MMP-13 level in SF was 1.333±0.6163ng/ml,which was higher than that in rats administered the HA2MRS(0.3654±0.3703 ng/ml),rats administered the LA2MRS(0.7051±0.3926 ng/ml),and rats that underwent sham operation(0.6763±0.2677 ng/ml).Conclusions 1)A2M as biomarker in SF could been adopted to early diagnosis PTOA and to monitor the degeneration of cartilage.It was critical to administ intraarticular A2 M injection due to rapidly decrease of A2 M in SF after operation.2)A2MRS with high protein active and high protein concentration could been made by ultrafiltration centrifugation.3)Supplemental intraarticular injection of A2 MRS attenuated cartilage degeneration in a rat model of ACLT,suggesting that it may be a potential novel therapy for PTOA. |
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