Advantages of Pure Platelet-Rich Plasma Compared with Leukocyte- and Platelet-Rich Plasma in Treating Rabbit Knee Osteoarthritis

Abstract

BACKGROUND Concentrated leukocytes in leukocyte- and platelet-rich plasma (L-PRP) may deliver increased levels of pro-inflammatory cytokines to activate the NF-κB signaling pathway, to counter the beneficial effects of growth factors on osteoarthritic cartilage. However, to date no relevant studies have substantiated that in vivo. MATERIAL AND METHODS Autologous L-PRP and pure platelet-rich plasma (P-PRP) were prepared, measured for componential composition, and injected intra-articularly after 4, 5, and 6 weeks post-anterior cruciate ligament transection. Caffeic acid phenethyl ester (CAPE) was injected intraperitoneally to inhibit NF-κB activation. All rabbits were sacrificed after 8 weeks postoperative. Enzyme-linked immunosorbent assays were performed to determine interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) concentrations in the synovial fluid, Indian ink staining was performed for gross morphological assessment, and hematoxylin and eosin staining and toluidine blue staining were performed for histological assessment. RESULTS Compared with L-PRP, P-PRP injections achieved better outcomes regarding the prevention of cartilage destruction, preservation of cartilaginous matrix, and reduction of IL-1β and PGE2 concentrations. CAPE injections reversed the increased IL-1β and PGE2 concentrations in the synovial fluid after L-PRP injections and improved the outcome of L-PRP injections to a level similar to P-PRP injections, while they had no influence on the therapeutic efficacy of P-PRP injections. CONCLUSIONS Concentrated leukocytes in L-PRP may release increased levels of pro-inflammatory cytokines to activate the NF-κB signaling pathway, to counter the beneficial effects of growth factors on osteoarthritic cartilage, and finally, result in a inferior efficacy of L-PRP to P-PRP for the treatment of osteoarthritis.

Figures

Figure 1

Study design. L-PRP – leukocyte- and platelet-rich plasma; P-PRP – pure platelet-rich plasma; CAPE – caffeic acid phenethyl ester.

Figure 2

Leukocyte and platelet concentrations in platelet-rich plasma formulations used in the study. (A) At each time point, the leukocyte concentrations in leukocyte- and platelet-rich plasma (L-PRP) used in the L-PRP group and L-PRP+ caffeic acid phenethyl ester (CAPE) group were higher than in pure platelet-rich plasma (P-PRP), which was used in the P-PRP group and P-PRP+CAPE group. (B) L-PRP and P-PRP were similar in platelet concentrations. Boxes and error bars represent mean ± standard deviation (n=10); * p<0.05 compared with L-PRP used in the L-PRP group at the same time point; #p<0.05 compared with L-PRP used in the L-PRP+CAPE group at the same time point.

Figure 3

Cytokine concentrations in platelet-rich plasma formulations used in the study. (A, B) At each time point, the concentrations of interleukin-1β (IL-1β); (A) Tumor necrosis factor-α (TNF-α). (B) Leukocyte- and platelet-rich plasma (L-PRP) used in the L-PRP group and L-PRP+ caffeic acid phenethyl ester (CAPE) group were higher than in pure platelet-rich plasma (P-PRP), which was used in the P-PRP group and P-PRP+CAPE group; (C, D) L-PRP and P-PRP were similar in the concentrations of platelet-derived growth factor AB (PDGF-AB). (C) Transforming growth factor β1 (TGF-β1). (D) Boxes and error bars represent mean ± standard deviation (n=10); * p<0.05 compared with L-PRP used in the L-PRP group at the same time point; #p<0.05 compared with L-PRP used in the L-PRP+CAPE group at the same time point.

Figure 4

Correlations between components of platelet-rich plasma formulations. There was a significantly positive correlation between leukocyte concentration and IL-1β concentration (A) and TNF-α (B) concentration, and between platelet concentration and PDGF-AB concentration (C) and TGF-β1 concentration (D). The correlations between leukocyte concentration and PDGF-AB (E) and TGF-β1 (F) concentrations and the correlations between platelet concentration and IL-1β (G) and TNF-α (H) concentrations were not significant. IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; PDGF-AB, platelet-derived growth factor AB; TGF-β1, transforming growth factor-β1.

Figure 5

Gross morphological assessment of cartilage degeneration. Gross morphological assessment was performed on both the medial and lateral sides of femoral condyles according to the following criteria after Indian ink staining: grade 1, no staining by Indian ink; grade 2, surface retains ink as elongated specks or light gray patches; grade 3, surface retains ink as intense black patches; grade 4, loss of cartilage exposing the sub-cartilaginous bone; grade 4a, 0 mm p<0.05 compared with the control group; #p<0.05 compared with the L-PRP group (n=40).

Figure 6

Interleukin 1β (IL-1β) and prostaglandin E2 (PGE2) concentrations in the synovial fluid. IL-1β (A) and PGE2 (B) concentrations in the synovial fluid were quantified by enzyme-linked immunosorbent assay. The mean IL-1β and PGE2 concentrations in the synovial fluid collected from rabbits of the L-PRP group were significantly higher than the control group, which, in turn, was significantly higher than the P-PRP group, L-PRP+CAPE group, and P-PRP+CAPE group, which were similar compared with each other. L-PRP, leukocyte- and platelet-rich plasma; P-PRP, pure platelet-rich plasma; CAPE, caffeic acid phenethyl ester. Boxes and error bars represent mean ± standard deviation (n=20); * p<0.05 compared with the control group; #p<0.05 compared with the L-PRP group.

Figure 7

Hematoxylin and eosin staining and toluidine blue staining for histological assessment. Representative HE stained sections (A, C, E, G, I) and toluidine blue stained sections (B, D, F, H, J) of femoral condyles after 8 weeks postoperative. L-PRP – leukocyte- and platelet-rich plasma; P-PRP – pure platelet-rich plasma; CAPE – caffeic acid phenethyl ester. Scales represent 200 μm.