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While it has been proved that centrifugal conditions for pure platelet-rich plasma (P-PRP) preparation influence the cellular composition of P-PRP obtained, the optimal centrifugal conditions to prepare P-PRP have not yet been identified. In the present study, platelet-containing plasma (PCP) was prepared with the first-spin of different double-spin methods and P-PRP was prepared with different double-spin methods. Whole-blood analysis was performed to evaluate the cellular composition of PCP and P-PRP. The basal and ADP-induced CD62P expression rates of platelets were assessed by flow cytometry to evaluate the function of platelets in PCP and P-PRP. Enzyme-linked immune sorbent assay was performed to quantify interleukin-1β, tumor necrosis factor-α, platelet-derived growth factor AB and transforming growth factor β1 concentrations of PCP and P-PRP. Correlations between the cellular characteristics and cytokine concentrations of P-PRP were analyzed by Pearson correlation analysis. Effects of P-PRP on the proliferation, survival and migration of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes were evaluated by a Cell Counting Kit-8 assay, live/dead staining and Transwell assay, respectively. The results showed that centrifugation at 160 x g for 10 min and 250 x g for 15 min successively captured and concentrated platelets and growth factors significantly more efficiently with preservation of platelet function compared with other conditions (P<0.05). The correlation analysis showed that the similar leukocyte concentrations and leukocyte-reducing efficiencies resulted in similar pro-inflammatory cytokine concentrations in P-PRP (P>0.05) and the maximization of platelet concentration, platelet enrichment factor, platelet capture efficiency and platelet function resulted in the maximization of growth factor concentrations in P-PRP obtained using the optimal conditions (P<0.05). Compared with P-PRP obtained under other conditions, P-PRP obtained under the optimal conditions significantly promoted the proliferation and migration of cells (P<0.05) and did not alter cell survival (P>0.05). Therefore, centrifugation at 160 x g for 10 min and 250 x g for 15 min successively with removal of the buffy coat as a crucial step may provide an optimal preparation system of P-PRP for clinical application. [ABSTRACT FROM AUTHOR]

Erythrocyte sedimentation rate (ESR), which reflects the sedimentation rate of platelets, leukocytes and erythrocytes in response to centrifugal force, may influence the cellular composition of platelet-rich plasma (PRP) obtained via centrifugation methods. However, no relevant studies have substantiated this. In the present study, blood was collected from 40 healthy volunteers and used to prepare PRP with two plasma-based preparation systems [YinPRP and Plasma Rich in Growth Factor (PRGF) systems] and two buffy coat-based systems (RegenPRP and WEGOPRP systems) in a single-donor model. Volumes of PRP and platelet-poor plasma (PPP) that were removed in the preparation process were recorded. Analyses of ESR, haematocrit, C-reaction protein, coagulation, serum glucose and serum lipid of the whole blood used for PRP preparation were performed to evaluate the levels of ESR and the factors known to influence it. Whole blood analysis was performed to evaluate the cellular composition of PRP. Results demonstrated that there were marked positive correlations between the ESR of the whole blood used for PRP preparation and PPP removal efficiencies, platelet concentrations, platelet capture efficiencies and platelet enrichment factors of PRP formulations obtained from plasma-based systems, and PRP yield efficiency of RegenPRP and PPP removal efficiency of WEGOPRP. Furthermore, there were marked negative correlations between ESR and concentrations and enrichment factors of platelets, leukocytes and erythrocytes of RegenPRP. Fibrinogen concentration of the whole blood, which had a marked positive correlation with ESR, also influenced the cellular composition of PRP. These findings may increase the understanding of PRP preparation and provide substantial evidence for the individualised optimisation of PRP preparation systems used in clinical practice. [ABSTRACT FROM AUTHOR]

Concentrated leukocytes in leukocyte and platelet-rich plasma (L-PRP) may deliver increased levels of pro-inflammatory cytokines to activate the nuclear factor (NF)-κB signaling pathway, to counter or overwhelm the beneficial effects of growth factors on cartilage regeneration. However, to date, no relevant studies have substantiated this. In the present study, L-PRP and pure platelet-rich plasma (P-PRP) were prepared, and leukocytes, platelets, pro-inflammatory cytokines and growth factor concentrations were quantified; they were then used to treat human articular chondrocytes (HACs). Pyrrolidine dithiocarbamate (PDTC; 50 μM) was used to inhibit the activation of NF-κB. The nuclear translocation of NF-κB p65 and the protein expression of cartilaginous markers (collagen II, aggrecan and sex-determining region Y-box 9) were determined using western blot analysis. The mRNA expression of NF-κB-dependent inflammatory mediators, including inducible nitric oxide synthase and cyclooxygenase-2, and cartilaginous markers were determined using reverse transcription-quantitative polymerase chain reaction analysis. The production of prostaglandin E2, nitric oxide and glycosaminoglycan (GAG) were quantified using enzyme-linked immunosorbent assays, the Griess reaction and a 1,9-dimethylmethylene blue assay, respectively. The results demonstrated that L-PRP induced the nuclear translocation of NF-κB p65, upregulated the mRNA expression of NF-κB-dependent inflammatory mediators and upregulated the production of their products, whereas P-PRP, which had similar growth factor concentrations but significantly lower pro-inflammatory cytokine concentrations than L-PRP, did not. P-PRP promoted the mRNA and protein expression levels of cartilaginous markers and the production of GAG more effectively, compared with L-PRP. Furthermore, inhibition of the activation of NF-κB by PDTC enhanced the effects of L-PRP on extracellular matrix formation in the HACs to a level similar to that of P-PRP. These findings suggested that leukocytes in L-PRP activated the NF-κB signaling pathway via the delivery of interleukin-1β and tumor necrosis factor-a to counter the beneficial effects of growth factors on extracellular matrix formation in HACs. Therefore, P-PRP may be more suitable for the treatment of osteoarthritis. [ABSTRACT FROM AUTHOR]