Customized Platelet Rich Plasma And Its Application In Tendon Repair

Objective:To identify a more popularized preparation protocol of leukocyte-rich platelet-rich plasma and the effect of growth factors released by platelets selectively activated via different receptors on the membrane of platelets on rats tendon stem cells（TSCs）and human umbilical vein endothelial cells（HUVECs）in proliferation,migration and differentiation.Methods:Twice centrifugations were taken.45mL peripheral blood collected from 76 volunteers was divided into three groups,mixed with5mL sodium citrate injection for transfusion,which are group A,400×g,10minutes for the first time and 1100×g,10 minutes for the second time,12samples included,group B,800×g,10 minutes for the first time and 1100×g,10 minutes for the second time,27 samples included,and control,1360×g,10 minutes for the first time and 1360×g,10 minutes for the second time,37samples included.Platelet recovery rate and enrichment factor were tested and evaluated.This protocol was used for preparation of rats’PRP whose enrichment factor and recovery rate were tested and evaluated.The centrifugal force and time of the first and second centrifugation of P-PRP was adjusted,and peripheral blood was collected from 28 volunteers（45 ml,mixed with 5 ml sodium citrate injection for blood transfusion）to test the most suitable centrifugal conditions of P-PRP,260g,10min,360g,15min,obtained in the research.Rats’TSCs and HUVECs were incubated in 37℃,5%CO₂ with supernatant separated from PRP activated via TFLLR-NH2（group PAR1）,AYPGK-NH2（group PAR4）and thrombin（group thrombin）after centrifugation,of which the proliferation and migration were measured.The expression of Oct4,Nanog,Scx,Collagen Iα1,DCN,TNC,PPARγ,Sox9,Collagen II and Runx2 were analyzed via quantitative real-time PCR.TSCs’adipogenic differentiation and osteogenic differentiation was observed by Oil Red O stain and Alizarin Red S.Results:55 samples were contained,which 10 samples were included in group A,21 in group B and 24 in control,after abnormal samples,which showed an exceeded platelet recovery rate higher than 100%or white thrombus,being removed.Our results revealed that significant differences of enrichment factor and recovery of platelets were shown between group B and group A and control,but no significant differences were shown between group A and control.There’re no significant differences between group A,B and control in the enrichment factor of leukocytes.Platelet recovery of P-PRP centrifuged via 260g、10min,360g、15min was 52.30±15.72%,while the enrichment of platelets and leukocytes was 5.230±1.572 and1.145±0.650 respectively.PRP selectively activated via PAR1 demonstrates its more significant role in the promotion of proliferation and migration of TSCs and HUVECs,while PRP selectively activated via PAR4 shows the reversed result.4 days later,proliferation of TSCs slowed down in all groups.It was a significant trend that PRP selectively activated via PAR1 propelled the adipogenic and chondrogenic differentiation of tendon stem cells,and the former will be demonstrated at early stage（7 days）.PRP selectively activated via PAR4 demonstrated a significant effect on preventing TSCs from differentiation.Meanwhile,PRP selectively activated by thrombin showed the inhibition of normal differentiation of TSCs.Moreover,TSCs in PAR1 group were Oil Red O-positive 14 days later.Conclusion:Our research confirms that it is an ideal and practical centrifugal condition for L-PRP,800×g,10 minutes for the first time and1100×g,10 minutes for the second time,while 260g,10 minutes for the first time and 360×g,15 minutes for the second time for P-PRP.PRP selectively activated via PAR1 could be chosen to improve the proliferation,migration of TSCs and vascularization of injured tendons within 24-48 hours（early stage）after tendon injury,while PRP selectively activated via PAR4 could be injected to the injured tendons to inhibit differentiation of TSCs and vascularization of injured tendons and to prevent tendinopathy 5-6 weeks（late stage）later.