Application Of Autologous Platelet-rich Plasma And Bone Marrow Concentrate In A Goat Model Of Osteoarthritis

Part oneProduction and evaluation of autologous platelet-rich plasma and bone marrow concentrateObjectiveTo product autologous platelet-rich plasma(PRP)and bone marrow concentrate(BMC)from whole blood(BM)and bone marrow aspirate(BM)by two-step centrifugation assay kits and evaluation the platelet,monocyte and mesenchymal stem cells(MSCs)concentrations and cytokines levels by physical and chemical methods.Methods(1)Production of PRPUnder general anesthesia,40ml whole blood was collected from the auricular vein and anticoagulated with acid citrate dextrose.PRP was produced by following a two-step centrifugation procedure under sterile conditions.The first step involved centrifugation at 350×g for 10 mins,following which the upper plasma layer and middle buffy coat were collected and subjected to a second centrifugation at 450×g for 15 mins to obtain 5 m L PRP.(2)Production of BMCIn a similar manner to the preparation of PRP,40 m L bone marrow(BM)was aspirated from both iliac crests and a two-step centrifugation procedure was used to produce 5 m L BMC.(3)DetectionsCellular constructions of PRP,WB,BM and BMC were observed afer centrifugation.The levels of TGF-?1 and IGF-1 in samples a were analyzed using ELISA kits.MSCs concentrations in BM and BMC were detected by flow cytometry following CD34 and CD44 marking.ResultsCompared with WB and BM,obvious while sediments were observed between plasma and erythrocyte layers.The concentration of platelets was 2.9-fold higher in PRP(1330.1×10~3/?L)than WB(461.7×10~3/?L)(P<0.05).Platelets numbers were also found to be 1.8-fold higher in BMC(1190.3×10~3/?L)compared to BM(663.2×10~3/?L)samples(P<0.05).However,no significant difference was observed between PRP and BMC platelet counts.The concentration of monocytes was 5.6-fold and 10.5-fold higher in samples of BMC(2.117×10~3/?L)than BM(0.378×10~3/?L)and PRP(0.202×10~3/?L),respectively(P<0.05).The level of TGF-?1 was found to be higher in PRP(7.308 ng/m L)than WB(2.232 ng/m L).It was also shown to be higher in BMC(14.621 ng/m L)than BM(1.912 ng/m L),and in BMC than PRP(P<0.05).IGF-1 levels in BMC(110.3 ng/m L)were 3.6-fold and 3.1-fold greater than BM(30.28 ng/m L)and PRP(35.14 ng/m L),respectively(P<0.05).The rates of MSCs of CD34(+)and CD44(+)were significant elevated in BMC than BM by flow cytometry.ConclusionAfter the two-step centrifugation,the platelet concentration and TGF-?1 level in PRP were increased than WB,and the platelet,monocyte and MSCs concentrations and TGF-?1 and IGF-1 levels in BMC were elevated than BM.It shows that the production procedures of autologous PRP and BMC with two-step centrifugation assay kits were reliable and feasible.Part twoIntraarticular injection autologous platelet-rich plasma and bone marrow concentrate in a goat osteoarthritis modelObjectiveAutologous biomaterials have been investigated as novel treatments for osteoarthritis.This study was designed to evaluate the effects of intraarticular injections of autologous platelet-rich plasma(PRP)or bone marrow concentrate(BMC)in a goat osteoarthritis(OA)model.MethodsTwenty-four adult goats were randomly divided into control(Ctrl),saline(NS),PRP,and BMC groups(n = 6).An OA model was induced by surgical anterior cruciate ligament transection and medial meniscectomy in NS,PRP,and BMC groups.After 6 weeks,PRP and BMC were obtained following a two-step centrifugation procedure from whole blood and bone marrow,respectively.Equivalent amounts of saline,PRP and BMC were administered by intraarticular injection once every four weeks,three consecutive times.Six weeks after the final injection all animals were sacrificed and the synovial fluid in samples were collected for general observation,and the concentrations of interleukin-1?(IL-1?),interleukin-6(IL-6),tumor necrosis factor-?(TNF-?)and prostaglandin E2(PGE2)in the synovial fluid were measured using ELISA kits.The progression of bone and cartilage degeneration was observed by macroscopy,histology,and immunohistochemistry.OARSI and modified Markin scores were performed to evaluate the macroscopy and pathology changes in samples.ResultsThe appearance of synovial fluid in the control group is yellowish and transparent.The joint fluid of NS group was hemorrhagic,and more floccus was seen.The joint fluid of PRP group and BMC group was pale red,and the floccus was significantly reduced compared with NS group.After centrifugation,the platelet and TGF-?1 content of PRP and BMC samples was increased(P < 0.05).Compared with PRP,higher levels of monocytes,TGF-?1,and IGF-1were observed in BMC(P < 0.05).Compared with the NS group,the synovial levels of IL-1?,IL-6,TNF-?,and PGE2 were lower in the BMC group,whereas IL-6 and PGE2 levels were found to be reduced in the PRP group(P < 0.05).The level of IL-1? was found to be lower in the BMC group than the PRP group(P < 0.05).Histologically,compared with the NS group,delayed cartilage degeneration and higher levels of extracellular matrix(ECM)were observed in both PRP and BMC treated groups.Furthermore,the BMC group showed greater cartilage protection and less ECM loss than the PRP group.ConclusionIntraarticular injection of autologous PRP and BMC showed therapeutic efficacy in a goat osteoarthritis model.The BMC-treated group demonstrated abundant monocytes and cytokines,and superior cartilage protection,suggesting that BMC may be an effective autologous biomaterial for treatment of OA.