Effect And Mechanism Study Of Exosomes Derived From Platelet-rich Plasma In The Treatment Of Knee Osteoarthritis

Objective:The purpose of this study was to extract autogenous platelet-rich plasma from New Zealand white rabbits by two-step centrifugation,and to study the exosomes secreted by platelet-rich plasma.Furthermore,the exosomes derived from platelet-rich plasma were analyzed and identified.The effect of platelet-rich plasma derived exosomes on the proliferation and apoptosis of osteoarthritis chondrocytes in vitro and its related signaling pathways were further explored.The therapeutic effect of exosomes derived from platelet-rich plasma on osteoarthritis in animal models was observed.These experiments would provide a firm theoretical support and evidence-based medicine basis for the early treatment of osteoarthritis with an effective treatment and the transformation of clinical results of PRP-exos.Method:First part:Blood was extracted from the ear vein of New Zealand white rabbits and platelet-rich plasma(PRP)was extracted by two-step centrifugation.Then,exosomes derived from platelet-rich plasma(PRP-exos)were extracted by "exoEasy Maxi Kit".The exosomes were observe the morphology by transmission electron microscope(TEM)and estimated the size and concentration distribution by Particle Metrix ZetaViewR Nanoparticle Tracking Analysis(NTA)technology.And western blotting was used to examine the specific exosome biomarkers.The exosomes were stored at-80°.Second part:After stripping the cartilage layer from both knees of the New Zealand white rabbit and adding collagenase,primary chondrocytes were isolated and extracted,then were identified by Alicin blue,SafraninO-Fast Green Staining and immunohistochemical staining(COL ?),further cultured for other experiments(within three generations).IL-1?(lOng/ml)was added to the P1 chondrocytes to establish chondrocytes model in osteoarthritis.Enzyme linked immunosorbent assay(ELISA)was used to measure the expression of TNF-?.The groups according to the experimental design are as follows?Control group,?IL-1? group,?IL-1?+PRP-as(5ug/ml)group,?IL-1?+PRP-as(50ug/ml)group,?IL-1?+PRP-exos(5ug/ml)group,?IL-1?+PRP-exos(50ug/ml)group.Flow cytometry,cell proliferation test(CCK-8),migration test and scratch test were used to compare the effects of PRP-exos with activated platelet-rich plasma(PRP-as)to compare the effect of PRP-exos and PRP-as on proliferation and apoptosis of osteoarthritis chondrocytes.The expression of ?-catenin,RUNX2 and Wnt5a in osteoarthritis chondrocytes after co-culturing with PRP-exos and PRP-as was determined by Western Blotting to investigate the relevant signaling pathway Third Part:Forty-two New Zealand white rabbits were randomly divided into Normal group,Sham group,Model group and experimental group.The experimental group was further divided into four subgroups:(1)PRP-as group(10ug/ml);(2)PRP-as group(100ug/ml);(3)PRP-exos group(10ug/ml);(4)PRP-exos group(100ug/m),sixe each group.The left knee joint of the normal group did not undergo any surgical treatment.In shaml group,only the left knee capsule was cut open without any other intra-articular treatment,and then was sutured.The left knee joint of the model group and the experimental group were surgeried with modified Hulth method to establish osteoarthritis model with resecting medial meniscus,cutting off medial collateral ligament and anterior cruciate ligament.Eight million units of penicillin were injected intramuscularly within 7 days to prevent infection after modeling.(1)Control group.Six weeks after modeling,10ml of autologous blood was extracted from the ear vein of each experimental group.Platelet-rich plasma and exosomes derived from platelet-rich plasma were prepared by the above methods.And left knee articular cavity injection was performed according to the concentration requirements of the experimental group.In the sham group and the model group,the same amount(0.6ml)of normal saline was injected into the joint cavity in the same way.Injection was given once a week,and each group was killed after 6 weeks of continuous injection.Gross morphological observation,hematoxylin eosin staining(HE staining),immunohistochemical staining(COL-2,RUNX2)and OARSI score were used to compare the efficacy of PRP-exos and PRP-as in the treatment of osteoarthritis.Result:First part:Platelet rich plasma was successfully isolated by two step centrifu--ge from Zealand white rabbit,and further treated by exoEasy Maxi Kit to extracte exosomes.Transmission electron microscopy(TEM)showed that PRP-exos is a round or elliptic bilayer of lipid vesicles.The diameter range was 145.6±50.4 an by using the Nanoparticle Tracking Analysis(NTA)technique.The expressions of CD9,CD63 and HSP101 on the surface of PRP-exos were determined by western blotting(WB).All of them are specific marker molecules of exosomes,and further suggests that the extract is exosomes.Second part:In this study,chondrocytes were successfully extracted and cultured,which were triangular,polygonal and spindle-shaped.The proteoglycan deposits of chondrocytes can be dyed blue by Alcian blue,and the cell matrix was dark red after being dealt with SafraninO-Fast Green Staining.Immunohistochemical staining(COL-?)was performed on the chondrocytes,presenting a blue nucleus and a tan cytoplasm.These staining results effectively proved that the extracted cells were chondrocytes.In vitro culture,this study found that IL-1?(10 ng/ml)could successfully induce chondrocyte inflammatory changes with increasing the release of TNF-?(OD value 2.58±0.11,P<0.05).After adding different concentrations of PRP-exos and PRP-as,OD value of TNF-? were significantly decreased(PRP-exos 5ug/ml:2.16±0.08;PRP-exos 50ug/ml:2.05±0.13;PRP-as 5ug/ml:2.25±0.06;PRP-as50ug/ml:2.08±0.04;P<0.05))were significantly decreased.However,there was no significant statistical difference between the groups(P>0.05).In vitro,chondrocytes in the induced group were stimulated by IL-1?(10 ng/ml),and the proliferation of osteoarthritis chondrocytes in the experimental group after adding PRP-exos and PRP-as at different concentrations(5ug/ml,50ug/ml)was significantly better than that in the induced group after being cultured with 24h,48h,72h and 120h in CCK8 test(P<0.05).The high concentration group of PRP-exos was better than the group of the PRP-as at all time periods(P<0.05),while PRP-exos was better than the PRP-as at 24h and 48h after cultured(P<0.05),and there was no statistical difference at 72h and 120h after cultured(P>0.05).Through apoptosis assay(flow cytometry),we found that high concentrations of PRP-exos and PRP-as significantly inhibited chondrocyte apoptosis induced by IL-1?(PRP-exos 50ug/ml:10.45±0.36;PRP-as 50ug/ml:12.34±0.42;induced group:13.85±0.34;P<0.05,and the effect of PRP-exos was significantly better than PRP-as with statistical difference(P<0.05).However,in the low concentration group,the inhibitory effect of the two on chondrocyte apoptosis was not statistical difference(P>0.05).In cell migration assay,we found that IL-1?(10 ng/ml)significantly inhibited chondrocyte migration.However,both PRP-exos and PRP-as can significantly promote chondrocyte migration.And the effect of the former is greater than that of the latter at the same concentration,and the effect of high concentration of PRP-exos and PRP-as was greater than that of low concentration group at the same time.Cell scratch assay showed that only high concentration of PRP-exos could promote the obvious migration of chondrocytes after continuous culture for 6h and 12h(P<0.05),while the migration of chondrocytes in other groups had not statistically significant(P>0.05).In the study of related signaling pathways by Western blotting,we found that osteoarthritis chondrocytes by IL-1? induced were highly expressed in ?-catenin,RUNX2 and Wnt5a in the Wnt/?-catenin signaling pathway.However,the expression of them could be decreased after the addition of PRP-exos and PRP-as,which had significant statistical significance(P<0.05).The decreased effect of RUNX2 and Wnt5a caused by PRP-exos was greater than that by PRP-as(P<0.05),while the decreased effect of ?-catenin expression was less(P<0.05).Third part:No accidental deaths occurred in this animal study.The osteoarthritis model of left knee joint in New Zealand white rabbits was successfully constructed by modified Hulth method and randomized controlled group experiment was conducted.After 6 weeks of modeling,the experimental animals were killed by air embolism,and the specimens were obtained.According to gross morphological findings,the knee joint of the model group,namely the OA group,presented that synovial tissue was edema,the cartilage of the femoral internal condyle presented a dull gray color,the touching surface was uneven,the visible part of the femoral internal condyle became thinner,and multiple osteophytes formed around the condyle.However,osteoarthritis changes in the PRP-exos group and PRP-as group were less severe than those in the model group.By HE staining,it was found that the cartilage surface of the model group was locally proliferated,the chondrocytes were irregularly arranged and the boundary was blurred,while the PRP-exos and PRP-as groups were significantly improved.The articular cartilage surface was still intact,the color was normal compared with that of the OA group,and there was no articular cartilage defect.Further chondrocyte counts showed that both PRP-exos group and PRP-as group could significantly increase chondrocytes(P<0.05),and the effect of PRP-exos was greater than that of PRP-as group(P<0.05).By immunohistochemical staining of the slices with COL II and RUNX2,We found that both PRP-exos and PRP-as could reverse the decreased expression of type II collagen caused by osteoarthritis and inhibit the expression of RUNX2 protein to promote cartilage repair,and the former had a greater effect than the latter(P<0.05).According to the score recommended by OARSI,the score of OA group was significantly higher than that of control group(P<0.05),while the score of PRP-exos group and PRP-as were lower than that of OA group,indicating that both PRP-exos and PRP-as could effectively inhibit osteoarthritis(P<0.05).Moreover,the score of PRP-exos was lower than that of PRP-as,indicating that the inhibitory effect of PRP-exos on osteoarthritis was greater than that of PRP-as(P<0.05).Conclusion:In this study,platelet-rich plasma(PRP)was successfully extracted from the blood of New Zealand white rabbits,then exosomes derived from platelet-rich plasma were identified and analyzed.At the same time,rabbit chondrocytes were successfully extracted and induced by IL-1? in vitro to establish a chondrocyte model for osteoarthritis.Studies had shown that PRP-exos could promote the proliferation and migration of osteoarthritis chondrocytes induced by IL-1? and inhibit chondrocyte apoptosis.PRP-exos may inhibit osteoarthritis through Wnt/?-catenin signaling pathway with the related protein expression and negative expression.Because the most pronounced effect of PRP-Exos was better than that of PRP,or some effects were similar,PRP-Exos may play the dominating role in the progress of PRP alleviating OA.In conclusion,we believe that exosomes derived from platelet-rich plasma(PRP-exos)have a potential role in the treatment of osteoarthritis,and intra-articular injection of PRP-exos can provide a novel approach for future exploration and application in clinical practice for the treatment of OA.