Platelet-Rich Plasma and Bone Defect Healing Angad Malhotra, PhD, Matthew Pelletier, PhD, Rema Oliver, PhD, Chris Christou, BScVet, and William R. Walsh, PhD On activation, platelets secrete an array of growth factors that contribute to bone regeneration. Combining platelet-rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autografts. Lack of standardization and improper use may contribute to the conflicting outcomes reported within both preclinical and clinical investigations using PRP. This study investigates the effect of PRP dose on bone augmentation. Eighty critical-sized defects were created in the cancellous bone of the medial proximal tibia and the distal femur of 20 five-year-old female sheep. The defects were treated with three doses of an autologous thrombin-activated PRP combined with a biphasic calcium phosphate (BCP) or autograft and empty defects. Radiography, micro-computed tomography, histology, histomorphometry, and fluorochrome bone labels were examined at 4 weeks. The empty defects did not spontaneously heal. The highest dose of PRP treatment had a significantly greater micro-CT bone volume/total volume compared with the BCP alone (PRP: 30.6% – 1.8%; BCP: 24.5% – 0.1%). All doses of PRP treatment were significantly greater than the BCP alone for histo- morphometric new bone area (PRP: 14.5% – 1.3%; BCP: 9.7% – 1.5%) and bone ingrowth depth (PRP: 2288 – 210 mm; BCP:1151 – 268 mm). From week 2 onward, PRP had a significant effect on the weekly bone ingrowth compared with BCP; however, autografts had the highest amount of weekly fluorescent bone labeling. PRP induces new bone formation with a dose-dependent response at 4 weeks when used with a BCP in sheep. Introduction B one grafting is an essential surgical tool for the augmentation of bone defects. Autogenous bone graft remains the most reliable treatment option due to its ability to provide an osteoconductive matrix, osteoinductive fac- tors, and osteogenic properties. 1–3 However, in addition to the limited supply, the harvesting of autograft has been as- sociated with a high rate of complications, in which *20% of patients suffer from donor-site morbidity and persistent pain, and *6% of cases require additional procedures. 3,4 Therefore, alternatives remain sought after. The basic elements for bone tissue engineering are signal- ing molecules, cells, and matrices. 5 During normal hemostatic mechanisms, a fibrin network is formed and platelets are ac- tivated, establishing the hematoma. 6 On activation, platelets release a variety of signaling molecules that orchestrate bone healing. 7,8 The basis of platelet-rich plasma (PRP) relies on the ability to concentrate and activate platelets, and to deliver a supra-physiologic concentration of the released growth factors to a defect site. The separation of blood components for surgical application has a long history. The collection of fibrinogen to use as an intraoperative fibrin glue for topical hemostasis found appli- cations in many clinical settings. 9 While the advantages of a hemostatic and adhesive fibrin glue are known, in 1994, Tayapongsak et al. 10 reported that the formation of the fibrin matrix also supported mandibular bone regeneration. The identification of platelet-released growth factors led to the de- velopment and use of PRP, initially reported in 1998 by Marx as beneficial for use in bone regeneration of mandibular de- fects. 11 This positive finding led to an increased interest and use of PRP within the oral and maxillofacial surgical fields. 12–14 Since this early application during the 1990s, PRP has seen prolific use across an increasing variety of surgical fields, and it now includes applications ranging from soft tissue healing, 15,16 cosmetic surgery, 17,18 burns, 19 nervous tissue, 20,21 and chronic skin ulcers. 22 In spite of the increasing use, contrasting out- comes, 8,23,24 compounded with inconsistent terminology and descriptions, 25,26 have hindered the progression of PRP use. The present study evaluated the efficacy of activated PRP combined with a biphasic calcium phosphate (BCP) to treat established critical-sized cancellous bone defects in older-aged sheep. Three PRP dosages were compared with a constant graft volume to provide insights into the effect of the PRP to graft ratio. In addition, to appreciate the role of platelets and other plasma constituents, platelet-poor plasma (PPP) combined with the BCP, and the BCP alone, were evaluated as reference groups. Iliac crest autograft and empty defects provided the positive and negative control groups. Overall, this study was Surgical and Orthopedic Research Laboratories, Prince of Wales Clinical School, The University of New South Wales, Prince of Wales Hospital, Randwick, Australia. TISSUE ENGINEERING: Part A Volume 20, Numbers 19 and 20, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2013.0737 2614