Platelets and platelet-leukocyte gel (PLG)

Platelets are small discoid blood cells that are formed from megakaryocytes and synthesized in the bone marrow. The average platelet count is 1.5–3.0 × 10⁵ per ml circulating blood. Inside the platelet, a number of intracellular structures exist. One of these structures is the α-granule which contains, among other substances, platelet-derived growth factors. In the resting state, platelets are nonthrombogenic and require a trigger before they become an active player in hemostasis and wound healing. Thrombin is the most potent platelet activator, causing platelets to change their shape. Following activation, platelets will aggregate and release the granulous content via the open canalicular system into the extracellular environment (Fig 1.2.3-1), where they bind to specific receptors. Through intracellular tissue signaling, a number of pathways are triggered to initiate the healing process [1, 2]. Platelet-leukocyte gel (PLG) is prepared from freshly drawn autogenous whole blood. A unit of whole blood is anticoagulated and subsequently sequestered by special centrifugal techniques into platelet-leukocyte rich plasma (P-LRP), platelet-poor plasma (PPP), and erythrocyte concentrate. P-LRP consists of a high concentration of inactive platelets in liquid form, fibrinogen, and leukocytes. After mixing with thrombin the platelets will be activated and a sticky gel mass of platelet aggregate is formed. In order to prevent a possible transmission of disease and cross-reaction from antibodies, the authors recommend the use of autogenously prepared thrombin, instead of bovine thrombin. Generally, the platelet numbers found in PLG are 3–9 times higher than in native whole blood [2].

Apart from the platelets, several differentiated and nonactivated leukocytes are present in PLG and these are known for their host-defense mechanism actions against bacteria. The leukocyte cell count in PLG is 2–4 times greater than in whole blood [2].

Platelets in bone healing

In fracture repair and bone healing, platelets act as an exogenous source of growth factors and therefore stimulate the activity of bone cells, based on their particular relevance to bone growth [3, 4]. At bone fracture sites, during normal bone healing, platelets release their growth factors, in particular platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), epidermal growth factor (EGF) and other cytokines.

TGF-β is an important growth factor in bone healing, and present during the various healing phases. Furthermore, TGF-β is abundantly present in platelets, bone, and cartilage. Two isoforms are found in platelets: TGF-β1 and TGF-β2. Chondrocytes and osteoblasts are enriched with receptors for TGF-β1. Therefore, this isoform has the greatest potential for bone repair.

Furthermore, TGF-β plays an active role in the bone healing process since it is present during all bone healing stages [5]. Therefore, it is postulated that a high concentration of platelet growth factors play a key role in the signaling process for osteogenesis and osteoinduction, through an osteogenic cell response [6]. In addition, the platelet growth factors PDGF and TGF-β1 contribute significantly into the differentiation of mesenchymal stem cells (MSC) into mature cells [1].

PLG and bone grafting

Nonunions are characterized by bone defects, and bone grafts are widely used to overcome these defects to enhance fusion. It is hypothesized that mixing PLG with autogenous bone might create a bioengineered graft (Fig 1.2.4-2) [1]. This graft contains platelets, which release growth factors, thrombin, fibrin, and other cytokines. Furthermore, as a result of the viscous nature of this graft, the migration of bone particles is avoided, since they will stick to the graft as a result of fibrinogen activation. Mitogenic activity within this active graft will result, among others, in the aggregation of MSC. This process is induced by the high concentration of platelets. These MSCs then have the potential to ultimately differentiate into osteoblasts, and thereby contribute to bone regeneration [7]. The underlying principle of PLG application is to mimic and accelerate the natural healing process. Several animal studies showed a quantifiably improved bone growth when bone grafts were mixed with PLG [8, 9]. Kim et al and Zechner et al showed in their animal studies the benefit and potential of PLG for the treatment of bone defects around dental implants [10, 11].

At present, there is hardly any literature about the application of PLG for nonunions. Chiang et al conducted a study in 12 patients with nonunions of the lower extremity. Patients were treated with autogenous bone grafting, enriched with PLG [12]. The results of this small, nonrandomized study, showed successful healing of nonunions with the addition of PLG, but this effect could not completely be attributed to PLG. A study of Galasso et al in 22 patients with long bone nonunions demonstrated comparable healing rates with a lower complication rate [13]. Bielecki et al investigated the role of percutaneous injection of autogenous PLG in 32 patients with delayed bone healing and the development of a nonunion, with satisfactory results [14].

Regarding the principles of bone healing and the potential contribution of platelet leukocyte gel for the treatment in nonunions, PLG could be a useful tool in supporting treatment of nonunions, despite the fact that there is little evidence available. Therefore, the authors recommend randomized clinical trials to be initiated in order to investigate the exact role of PLG and bone in the treatment of non-unions.