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

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          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.

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          Most cited references50

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          Neutrophil granules: a library of innate immunity proteins.

          Gene expression profiling has revealed that circulating neutrophils rest between two major bursts of transcriptional and protein synthetic activities. The first occurs in the bone marrow. This equips the neutrophil with stocks of innate defense armory that are packaged into different granule subsets. The second burst occurs when the neutrophil exits circulation and migrates into tissues to find, capture and phagocytose microorganisms. This burst results in the synthesis and secretion of cytokines and chemokines that support resolution of inflammation and healing of damaged tissue. Gene expression profiling has revealed that neutrophils express a variety of innate immunity proteins, known previously only to be expressed in other cells. Likewise, it has become clear that some proteins previously thought to be specific to the neutrophil are expressed in epithelial cells during inflammation.
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            Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma.

            Previous studies of bioactive molecules in platelet-rich plasma (PRP) have documented growth factor concentrations that promote tissue healing. However, the effects of leukocytes and inflammatory molecules in PRP have not been defined. The hypothesis for this study was that the concentration of growth factors and catabolic cytokines would be dependent on the cellular composition of PRP. Controlled laboratory study. Platelet-rich plasma was made from 11 human volunteers using 2 commercial systems: Arthrex ACP (Autologous Conditioned Plasma) Double Syringe System (PRP-1), which concentrates platelets and minimizes leukocytes, and Biomet GPS III Mini Platelet Concentrate System (PRP-2), which concentrates both platelets and leukocytes. Transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-AB (PDGF-AB), matrix metalloproteinase-9 (MMP-9), and interleukin-1β (IL-1β) were measured with enzyme-linked immunosorbent assay (ELISA). The PRP-1 system consisted of concentrated platelets (1.99×) and diminished leukocytes (0.13×) compared with blood, while PRP-2 contained concentrated platelets (4.69×) and leukocytes (4.26×) compared with blood. Growth factors were significantly increased in PRP-2 compared with PRP-1 (TGF-β1: PRP-2 = 89 ng/mL, PRP-1 = 20 ng/mL, P < .05; PDGF-AB: PRP-2 = 22 ng/mL, PRP-1 = 6.4 ng/mL, P < .05). The PRP-1 system did not have a higher concentration of PDGF-AB compared with whole blood. Catabolic cytokines were significantly increased in PRP-2 compared with PRP-1 (MMP-9: PRP-2 = 222 ng/mL, PRP-1 = 40 ng/mL, P < .05; IL-1β: PRP-2 = 3.67 pg/mL, PRP-1 = 0.31 pg/mL, P < .05). Significant, positive correlations were found between TGF-β1 and platelets (r(2) = .75, P < .001), PDGF-AB and platelets (r(2) = .60, P < .001), MMP-9 and neutrophils (r(2) = .37, P < .001), IL-1β and neutrophils (r(2) = .73, P < .001), and IL-1β and monocytes (r(2) = .75, P < .001). Growth factor and catabolic cytokine concentrations were influenced by the cellular composition of PRP. Platelets increased anabolic signaling and, in contrast, leukocytes increased catabolic signaling molecules. Platelet-rich plasma products should be analyzed for content of platelets and leukocytes as both can influence the biologic effects of PRP. Depending on the clinical application, preparations of PRP should be considered based on their ability to concentrate platelets and leukocytes with sensitivity to pathologic conditions that will benefit most from increased platelet or reduced leukocyte concentration.
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              International Cartilage Repair Society (ICRS) and Oswestry macroscopic cartilage evaluation scores validated for use in Autologous Chondrocyte Implantation (ACI) and microfracture.

              For young patients with cartilage defects, the emergence of clinically applicable cell therapy for biological joint reconstruction is an appealing prospect. Acceptation of this method as a means of standard care requires proof of being reproducible, having long-lasting mechanical integrity, and having a good clinical outcome. This study evaluates the reliability of the International Cartilage Repair Society (ICRS) score and the Oswestry Arthroscopy Score (OAS) in the assessment of regenerative cartilage repair. A total of 101 macroscopic images of cartilage repair were made during arthroscopy 12 months post-treatment of either Autologous Chondrocyte Implantation (ACI) or microfracture. These images were examined by seven independent observers with differing levels of experience. The ICRS and OAS scores were randomly presented twice at a 4-week interval. All observers stated their predicted outcome according to actual treatment and defect size. ICRS and OAS scores showed both good inter- and intra observer reliability (0.62 and 0.56 for ICRS; 0.73 and 0.65 for OAS, respectively). Internal consistency (Cronbach's alpha) was satisfactory for research purposes (0.79 and 0.74, respectively). Correlation (equivalence concordance) between both scoring systems was excellent (r=0.94). All observers were inconsistent in predicting actual treatment. Test-re test reliability of estimated defect size and its correlation to true defect size were poor. These results were also applicable to the sub-analyses of the experience of the observer and the quality of imaging. The ICRS and OAS are reliable and relevant scores that are now both validated for macroscopic evaluation of cartilage repair as a research tool.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                07 March 2017
                2017
                : 7
                : 43301
                Affiliations
                [1 ]Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital , Shanghai, China
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                srep43301
                10.1038/srep43301
                5339695
                28265109
                f3d83f39-d38f-4c59-8f62-28c5bdfac181
                Copyright © 2017, The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 08 April 2016
                : 24 January 2017
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