Proliferation and Differentiation of Human Tenocytes in Response to Platelet Rich Plasma: An In Vitro and In Vivo Study Xiao Wang, 1 Yiwei Qiu, 1 James Triffitt, 1 Andrew Carr, 1 Zhidao Xia, 1,2 Afsie Sabokbar 1 1 Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Windmill Road, Oxford OX3 7LD, UK, 2 Swansea University, School of Medicine, Swansea, UK Received 6 May 2011; accepted 31 October 2011 Published online 18 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22016 ABSTRACT: Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstruc- tive surgeries. ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:982–990, 2012 Keywords: activated platelet rich plasma (PRCR); human tenocytes; proliferation and differentiation Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and in- sulin-like growth factor (IGF-1). 1 An important charac- teristic of autologous PRP is that it eliminates concerns about immune responses and disease trans- mission within the patient. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for several types of clinical applications, including wound healing in surgery 2–4 ; regeneration of bone 5–7 and disc cartilage, 8,9 spinal fusions, 10–12 and joint arthroplasty 13–15 and tendon regeneration. 16–18 Despite these promising results, there have been a number of reports indicating limited success rate in clinical trials. 19–23 The regenerative potential of PRP depends on, to a large extent, the production and release of bioactive factors after the activation of platelets hence a number of studies have considered the use of platelet rich plas- ma-clot release (PRCR) instead of PRP. 24–26 There is a limited number of in vitro investigations whereby PRCR were employed for regenerating human tendon. For instance, Anitua et al. 27 reported that 20% PRCR enhanced human tenocytes cell proliferation and levels of VEGF and hepatocyte growth factor (HGF) release by the tenocytes. 27 In another study, de Mos et al. 28 have indicated that it is possible to induce human tenocyte proliferation when combining 10% PRCR with 2% fetal bovine serum (FBS). Although these results are encouraging, the use of FBS with PRCR is not suitable for any future in vivo investigations due to any possible host-versus-graft immune responses. Furthermore, these investigators focused their studies on determining a few markers of tendon biology such as matrix metalloproteinases (MMPs) and collagen I and III (COL-I and -III). However, recent evidence suggests that tenocytes are capable of expressing other markers such as Scleraxis (SCX) and Decorin (DCN). These markers could be therefore employed to further elucidate the possibility of using PRCR in regenerating human tendon in vitro and in vivo. The aims of the present study were therefore to fully elucidate whether PRCR could influence (i) the rate of human tendon proliferation and differentiation in vitro in the absence of FBS and (ii) in vivo human tendon formation using diffusion chambers (DCs) in murine models. The main objective is therefore to demonstrate that PRCR could substitute conventional FBS for in vitro and in vivo investigation with the potential to employ PRCP for future tendon recon- structive surgery. This approach is likely to be supe- rior to the currently employed PRP in clinical surgery as the activation of platelets augments the process of healing and tissue regeneration. METHODS PRCR Preparations Fifty milliliters of whole blood from three healthy nonsmok- ing donors (demographic details listed in Table 1) were collected in citrate vacutainers (buffered sodium citrate, 0.109 M, 3.2%, BD, Oxford, UK) and processed within 8 h. Protocols for PRCR preparation was based on the previously described protocol. 27,28 Briefly, whole blood was centrifuged Correspondence to: Xiao Wang (T: þ44 (0) 01792 606829; F: þ44 (0) 1792 602147; E-mail: goodthing1209@gmail.com) ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 982 JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2012