Optimization of Leukocyte Concentration in Platelet-Rich Plasma for the Treatment of Tendinopathy

BACKGROUND:Numerous methods are available for platelet-rich plasma (PRP) generation, but evidence defining the optimum composition is lacking. We hypothesized that leukocyte-reduced PRP would result in lower inflammatory cytokine expression compared with concentrated-leukocyte PRP and that maintaining the platelet:white blood cell (WBC) ratio would compensate for the effect of increased WBC concentration. METHODS:Blood and flexor digitorum superficialis tendons were collected from young adult horses. Three PRP groups were generated with the same platelet concentration but different WBC concentrationsintermediate-concentration standard PRP, leukocyte-reduced PRP, and concentrated-leukocyte PRP. An additional high-concentration PRP group was generated with the same WBC concentration as the concentrated-leukocyte PRP group and the same platelet:WBC ratio as the standard PRP group. The PRP groups were used as media for flexor digitorum superficialis tendon explants in culture for seventy-two hours with 10% plasma in Dulbecco modified Eagle medium (DMEM) serving as control. Tendon gene expression for collagen types I (COL1A1) and III (COL3A1), cartilage oligomeric matrix protein (COMP), matrix metalloproteinase (MMP-13), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) was performed. RESULTS:The desired PRP groups were successfully generated. The expression of COMP, the COL1A1:COL3A1 ratio, and the expression of MMP-13 in flexor digitorum superficialis tendon explants was not different between PRP groups. The expression of COMP (p = 0.0027) and the COL1A1:COL3A1 ratio (p < 0.0001) were increased in the PRP groups as compared with the control group, and the expression of MMP-13 was decreased in the PRP groups as compared with the control group (p < 0.0001). The expression of IL-1β was lowest in leukocyte-reduced PRP and highest in concentrated-leukocyte PRP (p = 0.0001). The leukocyte-reduced PRP group and the control group had the lowest TNF-α expression, whereas the high-concentration PRP and concentrated-leukocyte PRP groups had the highest expression (p = 0.0224). CONCLUSIONS:A high absolute WBC concentration in PRP contributes to the expression of inflammatory cytokines in flexor digitorum superficialis tendon explants, and maintenance of the platelet:WBC ratio is not able to counteract this effect. CLINICAL RELEVANCE:The optimum composition of PRP for the treatment of tendinopathy has not been directly investigated. Persistent inflammation resul