*2.6. PRGF Preparation and Application*

Platelet-rich plasma (PRP) was prepared according to the PRGF-Endoret method. PRGF is a leukocyte- and erythrocyte-free PRP with moderate platelet-enrichment [7]. Briefly, 72 mL of peripheral venous blood was withdrawn into 9 mL tubes containing 3.8% (wt/vol) sodium citrate (Endoret Traumatology kit, BTI Biotechnology Institute, Vitoria, Spain) before starting the patient's anesthesia. Then, the blood was centrifuged for 8 min at 580 g at room temperature in a System V centrifuge (BTI Biotechnology Institute, Vitoria, Spain). After centrifugation, three layers were obtained (plasma, buffy coat and packed red blood cells). The upper layer of plasma (F1 fraction) was collected in order to prepare the PRGF membrane. The 2 mL plasma fraction located just above the buffy coat (F2 fraction) was collected, avoiding the leukocyte layer, and was used to perform infiltrations.

We performed intraosseous (4 mL into the ischial tuberosity once freshened) and intratendinous infiltrations (8 mL) of liquid PRGF into the suture-repair areas (Figure 6a) The F2 fraction was activated in a time-controlled way by the addition of PRGF activator (10% CaCl2) just before to these infiltrations performed with a 21 G − 0.8 × 40 mm needle. Finally, the sciatic nerve was wrapped with a PRGF membrane (Figure 6b) elaborated with 8 mL of liquid F1, which was activated with 160 μL of PRGF activator and maintained for at least 15 min at room temperature until the formation of a clot.

**Figure 6.** Application of PRGF. (**a**) Intratendinous infiltration of PRGF into the allograft suture-repair areas. (**b**) The sciatic nerve is wrapped with a PRGF membrane.

Furthermore, seven and fourteen days after the surgery, and assisted by US, we performed two infiltrations of PRGF in the sutured areas of the graft. In both cases, 8 mL of freshly activated F2 PRGF was infiltrated [4,5].
