Research On The Therapeutic Effect And Mechanism Of ?2MRS Autologous Joint Injection In Treating Post-traumatic Osteoarthritis In Minipigs

Background Post-traumatic osteoarthritis is a cartilage degenerative disease caused by knee ligament,meniscus or knee cartilage injury.The specific pathogenesis is still unknown.At present,it is believed that abnormal changes in biochemical and biomechanical may be the main causative factors of PTOA,but relative contribution is unknown.Although the idealized anterior cruciate ligaments reconstruction model successfully reproduces the pathological process of PTOA for human ACL-R.Whether the knee biomechanical are completely restored and the mechanical interference factors are completely ruled out,which requires further scientific research.Alpha-2 macroglobulin,a macromolecular glycoprotein in biological fluids,has a broad-spectrum protease inhibitory effect and special joint cartilage protection.With further research,the bottleneck problem of ?2M has been continuously highlighted,such as limited output and high cost,which restricts further research.platelet—rich plasma has been widely used in soft tissue diseases and proven to be effective.Inspired by it,exploring a simpler method of enriching serum ?2M and observing its effect on PTOA has a significant value.Objective: 1.Gait analysis was used to detect the gait parameters of knee joints in the mini-pig IACL-R model,and to evaluate its biomechanical recovery.2.Exploration of enriching serum ?2 macroglobulin,and the protein concentration and enrichment coefficient of ?2M were detected.3.Study on the effect and mechanism of ?2MRS autologous joint injection on Post-traumatic osteoarthritis in minipigsMethods: 1.Cytonics ultrafiltration centrifuge cup(100KD)was used to concentrate plasma with different centrifugal forces(3000g,4000 g,5000g)and different centrifugation times(20min,30 min,40min),and finally obtained the upper concentrated solution and the lower filtered solution.Enzyme-linked immunosorbent assay was used to detect ?2M concentration,Coomassie brilliant blue,and Western Blot were used to further verify the concentration and enrichment.2.Eighteen mature female minipigs(age,18 months)were randomized to three groups.The right hind foot underwent IACL-R.The left hind foot does nothing.Sham group: RH only underwent joint capsule incision,and saline was injected into the joint(n = 6)IACL-R group: RH underwent IACL-R,and saline was injected into the joint(n = 6)IACL-R + ?2MRS group:RH underwent IACL-R,and ?2MRS was injected into the joint(n = 6)Gait data were collected preoperatively(day 0)and postoperatively on days 7,15,30,45,60,75,and 90.Luminex Chip was used to detect inflammatory factors of synovial fluid preoperatively(day 0)and postoperatively on days 3,6,14,29,and 90.saline and ?2MRS were injected into the joint in different groups postoperatively on days 2,6,14,29.All animals were euthanized at 3 months after the surgery.Cartilage integrity of the right knee was evaluated using histology and imaging at 3 months postoperativelyResults: ?2M were enriched successfully from serum;The gait characteristics and trend of inflammatory cytokines in minipig IACL-R model were initially verified;The protective effect of ?2MRS on PTOA articular cartilage has been preliminary verified 1.Cytonics ultrafiltration centrifuge cup(100KD)can effectively enrich serum ?2Munder the conditions of 30 min and 5000 g.?2M concentration in serum(2.28 ± 0.26 mg / ml),in concentrated solution(11.13 ± 0.90 mg / ml),in filtered solution(0.000049 ± 0.00001 mg / ml).The recovery rate of ?2M is 97.66%,and the enrichment coefficient is 4.88.2.Gait analysis showed that the gait indexes of the Sham group mini-pigs recovered to the preoperative state 15 days after the operation.The IACL-R group returned to normal in 45 days after surgery,and deteriorated again in the later period(75 days).The IACL-R + ?2MRS group returned to normal after 30 days and remained until euthanasia,which the effect was significantly better than the IACL-R group.3.Luminex suspension chip showed that Interleukin(IL)changed significantly in all synovial fluid samples.Sham group increased early in the postoperative,and basically fell back to the initial level 4 weeks after surgery.The IACL-R group increased significantly in the early postoperative,decreased significantly in the later period(29 days postoperatively),and increased again in the final period(90 days postoperatively).The IACL-R + ?2MRS group have a relative high concentration in the early postoperative,dropped significantly in the middle period until euthanasia,which the concentration was significantly lower than the IACL-R group.4.The results of imaging examination(X-ray,3D reconstruction CT,MRI)were consistent.The PTOA performance of mini-pigs in the Sham group was Slight,IACL-R + ?2MRS group is moderate and the IACL-R group was severe.MRI examinations are more sensitive,with significant differences in scores.5.None of the minipigs in the sham group exhibited severe macroscopic cartilage degeneration.The cartilage surface of all compartments was flat,smooth,and milky white or occasionally red in the femoral condyle.The edges of the sham group were smooth.In contrast,all the animals in the IACL-R group showed macroscopic degeneration of the articular surfaces.Large erosions down to the subchondral bone were observed occasionally.Mild irregular protrusions were found on both sides of only the trochlea in the IACL-R group.IACL-R + ?2MRS group exhibited moderate macroscopic cartilage degeneration with surface roughening and fissures.6.In the microscopic histological assessment,the sham group,we observed a slight decrease in safranin O staining and slight surface fibrillation.In the IACL-R group,however,we observed a moderate increase in chondrocyte density,increased chondrocyte cloning,severely increased fibrillation,and occasional erosions to the subchondral bone.Inthe IACL-R + ?2MRS group,the thickness of cartilage was normal,the surface fibrosis was moderate,the number and shape of chondrocytes were normal,and the score was significantly lower than in the IACL-R group.7.Immunohistochemical staining showed that Col-II expression was significantly higher and MMP-13 expression was significantly lower in the sham group than in the IACL-R group.The IACL-R + ?2MRS group has a moderate degeneration,which was between the above two groups.The experimental results are conducive to the further clarification of the pathogenesis of PTOA and provide a theoretical basis for the further clinical transformation of ?2MRSConclusion: 1..Cytonics ultrafiltration centrifuge cup(100KD)can effectively enrich serum ?2M under the conditions of 30 min and 5000 g 2.Gait analysis system proved that the IACL-R model restored the normal biomechanics of the knee joint,which can be used to independently assess the role of inflammatory factors in the PTOA.3.Early injection of ?2MRS in the joint can significantly reduce the concentration of inflammatory factors,and has a positive therapeutic effect for articular cartilage degradation.