Effect Of Intra-articular Injection Of Autologous And Allogeneic Pure Platelet-rich Plasma On BMP-2 And Sox9 In Rabbit Knee Osteoarthritis Chondrocytes

Objective: In the early stage of Knee osteoarthritis(KOA),intra-articular injection of autologous,allogeneic pure platelet-rich plasma.The purpose of this study is preliminarily exploring the possible mechanism of P-PRP in the treatment of KOA,comparing the differences of KOA between the two P-PRP groups by pathological and immunohistochemical methods.Methods:(1)Preparation of P-PRP: Following the principle of secondary centrifugation,the first relatively low speed centrifugation(1050r/min,centrifugal radius 8cm),the most sedimentation coefficient of the red blood cells to the bottom of the tube after centrifugation.Second relatively high-speed centrifugation(2100r/min,centrifugal radius 8cm),serum platelets collected as much as possible to the bottom of the tube,with a small amount of plasma resuspended to obtain P-PRP.(2)Subjects and interventions: Thirty-four healthy New Zealand rabbits were purchased from the Animal Experiment Center of Southwest Medical University.Eight of them were randomly selected as the normal control group by random number table.The remaining 26 rabbits were intra-articular injected with 5% papain,0.03 mmol/L L-cysteine mixed solution(0.1 ml/kg)to establish KOA model.Two rabbits were randomly selected and sacrificed at 2 weeks after the first injection to observe the right knee joint disease.After establishing the model successfully,24 rabbits were divided into model group(n = 8),autologous P-PRP group(n = 8),allogenic P-PRP group(n = 8).The autologous P-PRP group and the allogeneic P-PRP group received 0.5 ml autologous P-PRP and allogeneic P-PRP injected into the right knee joint cavity,once every 2 weeks for 3 times.Normal control group and model group received no treatment.Two weeks after the last intervention,all the rabbits were sacrificed by air embolism,samples were taken and the experimental indexes of each group were observed.(3)Observations:(1)According to OARSI rating and Peng Taiping standard,gross changes of the cartilage,osteophytes and synovialis in the right knee of all rabbits were rated by macroscopic observation.(2)The medial condylar cartilage of the right knee joint was made into HE staining and sealed.The arrangement of chondrocytes,chondrocyte morphology,stromal staining and intactness of the tidal line were observed under the light microscope.At the same time,the cartilage sections were graded according to the modified Mankin’s score.(3)Semiquantitative analysis of immunohistochemistry: The expression of BMP-2,Sox9 in the medial femoral condyle of the right knee joint of each group was detected.The average optical density of BMP-2 and Sox9 in each group was calculated by Image Pro Plus 6.0,indicating their positive expression.(4)Statistical analysis:Using IBM SPSS Statistics 22.0 software for statistical analysis,the resulting measurement values were expressed as((?)±S)and M(P25,P75)depending on whether or not the normality is satisfied.P <0.05 was considered statistically significant.Results:(1)The gross specimen observation: Compared with the model group,the autologous P-PRP group was significantly improved in articular cartilage repair,osteophyte and synovial fluid formation(P<0.05),while the allogeneic P-PRP group showed no significant improvement in the gross cartilage morphology(P>0.05).(2)Histopathological score of HE staining: Compared with model group,Mankin’s score of autologous P-PRP group and allogeneic P-PRP group were improved(P<0.05),but improvement of autologous P-PRP was the most obvious.Moreover,Mankin’s score between autologous P-PRP and allogeneic P-PRP was statistically significant(P <0.05).(3)Immunohistochemical evaluation:(1)BMP-2 immunohistochemistry showed the low expression in the normal group,the lowest expression in the model group.The expression of autologous P-PRP group and allogeneic P-PRP group was higher than that of the model group(P<0.05),and the highest expression was in autologous P-PRP group.The difference between autologous P-PRP group and allogeneic P-PRP group was statistically significant(P< 0.05).(2)Sox9 immunohistochemistry showed the highest expression level in normal group and the lowest in model group,while the expression of P-PRP group and allogeneic P-PRP group was higher than that of model group(P = 0.001,P =0.036).There was no significant difference between the normal group,autologous P-PRP group and allogeneic P-PRP group(P> 0.05).Conclusions:(1)Intra-articular injection of autologous P-PRP can activate the expression of BMP-2 and Sox9 in chondrocytes,increase the proliferation of chondrocytes and promote cartilage repair,so as to achieve the purpose of treating KOA;(2)Intra-articular injection of autologous P-PRP for KOA,It is superior to allogeneic P-PRP in promoting cartilage repair,reducing osteophytes,and reducing synovial fluid.