The goal of this study was to investigate if the preparation of implantation site has an impact on bone formation inside tissue engineering scaffolds. For this purpose, two different drilling techniques were used to create a hole in distal femurs of rats before the insertion of a bone scaffold: a manually driven wood drill bit and an electrically driven metal drill bit. The size and the position of the hole were identical for the two cases. The bone volume, bone mineral density, and callus formation were assessed non-invasively using micro-CT tomography at several time points after implantation. The formation of bone and soft tissue inside scaffold were evaluated by histology. The bone structure around the holes made by the two techniques was compared ex vivo. The long-term study of bone formation showed that when a wood drill bit was used, the bone formation is accelerated by three weeks compared to when a metal drill bit was used. The ex vivo studies suggest that this result is due to the drilling methods differentially affecting the structure of the bone surrounding the generated defects.