The aim of this study was to investigate de novo bone formation following ectopic site implantation of bone substitutes covered by periosteum, with and without the application of autologous platelet-rich plasma (PRP). Twenty-four weeks after subcutaneous implantation of various bone substitutes (bovine hydroxyapatite (bHAP), phycogenic hydroxyapatite (pHAP), and bioglass (BG)) in 35 mini-pigs, bone regeneration rates were compared microradiographically and histologically. Without PRP, bHAP showed a mean de novo bone formation of 32.41%±29.99, in contrast to the other substitute materials where no mineralization could be detected. In combination with PRP, in the bHAP (63.61%±12.98; p±0.03) and pHAP (34.37±29.38; p=0.015) group, significantly higher de novo bone formation was ascertained than without PRP. No ossification could be detected in the BG group. In conclusion, bHAP and pHAP bone substitutes in combination with PRP showed a significant positive effect on periosteal cells by de novo bone formation after ectopic, subcutaneous, low-vascular site implantation.