There are no studies on the cellular activity in the early phase of biodegradation and bone healing of bone substitutes loaded with platelet factors (PLF). The purpose of this study was to evaluate the cellular effects of PLF in combination with nanoparticulate hydroxyapatite (HA) on the biodegradation and bone formation after 20 days. Autogenous PLFs were obtained by centrifugation of miniature pig blood samples and subsequent degranulation of platelets by calcium and thrombin. A cylindrical bone defect with a diameter of 8.9 mm was created in the distal femoral condyle of 20 miniature pigs. Four of the defects were left empty, 8 were filled with HA with loading and 8 with HA loaded with PLF. The distal femur was harvested after 20 days and TRAP-staining, cathepsin-K and CD44 staining and scanning electron microscopy were performed for cellular assessment of biodegradation was done. Histomorphometry of new bone formation and of biodegradation of HA material was performed. PLF loading of HA led to statistically significant more TRAP-positive cells with enhanced biodegradation of the nanoparticulate HA but no statistically enhanced new bone formation compared to unloaded HA. Furthermore, there was a higher number of CD44 and cathepsin-K positive cells by PLF-loading. In summary, PLF led to stimulation of the cellular process of the biodegradation of HA.