Comparative evaluation of leukocyte- and platelet-rich plasma and pure platelet-rich plasma for cartilage regeneration

Abstract

Platelet-rich plasma (PRP) has gained growing popularity in the treatment of articular cartilage lesions in the last decade. However, the potential harmful effects of leukocytes in PRP on cartilage regeneration have seldom been studied in vitro, and not at all in vivo yet. The objective of the present study is to compare the effects of leukocyte- and platelet-rich plasma (L-PRP) and pure platelet-rich plasma (P-PRP) on cartilage repair and NF-κB pathway, in order to explore the mechanism underlying the function of leukocytes in PRP in cartilage regeneration. The constituent analysis showed that P-PRP had significantly lower concentrations of leukocytes and pro-inflammatory cytokines compared with L-PRP. In addition, cell proliferation and differentiation assays indicated P-PRP promoted growth and chondrogenesis of rabbit bone marrow mesenchymal stem cells (rBMSC) significantly compared with L-PRP. Despite similarity in macroscopic appearance, the implantation of P-PRP combining rBMSC in vivo yielded better cartilage repair results than the L-PRP group based on histological examination. Importantly, the therapeutic effects of PRP on cartilage regeneration could be enhanced by removing leukocytes to avoid the activation of the NF-κB pathway. Thus, PRP without concentrated leukocytes may be more suitable for the treatment of articular cartilage lesions.

Figure 1. Cellular and cytokine concentrations in WB, L-PRP and P-PRP.

Concentrations of leukocytes (A), IL-1β (B), and TNF-α (C) in P-PRP were significantly lower than those in L-PRP and WB; concentrations of platelets (D), PDGF-AB (E), and TGF-β1 (F) in L-PRP and P-PRP were similar, but significantly higher than those in WB. *p < 0.01 compared with WB; ^$p < 0.01 L-PRP versus P-PRP; n = 10.

Figure 2. Correlations between cytokine concentrations and cellular components in WB, L-PRP and P-PRP.

Positive correlations were observed between the concentrations of leukocytes and those of IL-1β (A) and TNF-α (B), and between the concentrations of platelets and those of PDGF-AB (C) and TGF-β1 (D) in WB, L-PRP and P-PRP; n = 30.

Figure 3. PRPs promoted proliferation and chondrogenetic differentiation of rBMSC.

(A) CCK-8 assay showed that both L-PRP and P-PRP promoted rBMSC proliferation on day 3, 5 and 7 compared with FBS, but P-PRP showed greater effects on rBMSC proliferation on day 5 and 7 compared with L-PRP; (B) western blotting analysis revealed that P-PRP upregulated protein expression of SOX-9, Aggrecan, and Col II, and downregulated protein expression of Col I, compared with L-PRP and FBS; (C–F), qRT-PCR analysis demonstrated that both L-PRP and P-PRP upregulated mRNA expression of SOX-9, Aggrecan, Col II, and Col I compared with FBS, but P-PRP showed greater effects on mRNA expression of chondrogenic-related marker genes (SOX-9, Aggrecan, and Col II) and weaker effects on mRNA expression of osteogenic-related marker gene (Col I) compared with L-PRP. *p < 0.01 compared with WB; ^#p < 0.01 compared with CDK; ^$p < 0.01 L-PRP versus P-PRP; n = 3.

Figure 4. L-PRP induced the activation of NF-κB pathway.

(A) Immunofluorescence staining revealed that NF-κB p65 was observed in the nucleus in the L-PRP group, and in the cytoplasm in the P-PRP and FBS groups; (B) western blotting analysis showed that L-PRP upregulated protein expression of NF-κB p65 in the nucleus compared with P-PRP and FBS; (C and D) qRT-PCR analysis showed that the mRNA expression levels of iNOS and COX-2 were significantly higher in the L-PRP group than those in the P-PRP and FBS groups; (E and F) Production of PGE2 and NO was significantly enhanced in the L-PRP group in comparison with that in the P-PRP and FBS groups. *p < 0.01 compared with WB; ^$p < 0.01 L-PRP versus P-PRP; n = 3.

Figure 5. Effects of PRPs on cartilage repair in rabbits — gross observation.

Macroscopic appearance of the cartilage healing in the control, L-PRP and P-PRP groups at 6 weeks (A–C), and 12 weeks (D–F) after implantation. n = 6.

Figure 6. Effects of PRPs on osteochondral repair in rabbits — quantitative analysis of regenerated bone tissue using a 3-D Micro-CT model.

(A) Region of interest (ROI) corresponding to the original defects in rabbits. The green colour represents regenerated bone, and the yellow colour represents other tissues in the original defect (top: 3-D models in situ; bottom: 3-D models ex situ). (B) Comparison of the bone regeneration in the 4 groups using the ratio of BV/TV, trabecular number, trabecular pattern factor, structure model index and connectivity density at 12 weeks. Non-treated rabbits were used as a positive control. BV, bone volume; TV, tissue volume; *p < 0.05 compared with the control group; ^#p < 0.05 compared with the positive control group; n = 6.

Figure 7. Effects of PRPs on cartilage repair in rabbits — H&E staining.

Representative H&E stained sections in the control, L-PRP and P-PRP groups at 6 weeks (A–F) and 12 weeks (G–L). The rectangular squares demonstrated the conjunction between the regenerated tissue (right half) and the normal tissue (left half). n = 6.

Figure 8. Effects of PRPs on cartilage repair in rabbits —toluidine blue staining.

Representative toluidine blue stained sections in the control, L-PRP and P-PRP groups at 6 weeks (A–F) and 12 weeks (G–L). The rectangular squares demonstrated the conjunction between the regenerated tissue (right half) and the normal tissue (left half). n = 6.

Figure 9. Effects of PRPs on cartilage repair in rabbits — immunohistochemical staining.

Representative sections using Col I (A–F) and Col II (G–L) staining in the control, L-PRP and P-PRP groups at 12 weeks. The rectangular squares demonstrated the conjunction between the regenerated tissue (right half) and the normal tissue (left half). n = 6.

Figure 10. Effects of PRPs on cartilage repair in rabbits — macroscopic and histological scoring.

(A) macroscopic ICRS scores; (B) histological OARSI scores. *p < 0.05 compared with the control group; ^$p < 0.05 L-PRP versus P-PRP; n = 6.