The aim of this study was to present new a model that allows the study of the bone healing process, with an emphasis on the biological behavior of different graft-to-host interfaces. A standardized “over-inlay” surgical technique combined with a differential histomorphometric analysis is presented in order to optimize the use of critical-size calvarial defects in pre-clinical testing.

Critical-size defects were created into the parietal bone of 8 male Wistar rats. Deproteinized bovine bone (DBBM) blocks were inserted into the defects, so that part of the block was included within the calvarial thickness and part exceeded the calvarial height (an “over-inlay” graft). All animals were sacrificed at 1 or 3 months. Histomorphometric and immunohistochemical evaluation was carried out within distinct regions of interest (ROIs): the areas adjacent to the native bone (BA), the periosteal area (PA) and the central area (CA).

The animals healed without complications. Differential morphometry allowed the examination of the tissue composition within distinct regions: the BA presented consistent amounts of new bone formation (NB), which increased over time (24.53%±1.26% at 1 month; 37.73%±0.39% at 3 months), thus suggesting that this area makes a substantial contribution toward NB. The PA was mainly composed of fibrous tissue (71.16%±8.06% and 78.30%±2.67%, respectively), while the CA showed high amounts of DBBM at both time points (78.30%±2.67% and 74.68%±1.07%, respectively), demonstrating a slow remodeling process. Blood vessels revealed a progressive migration from the interface with native bone toward the central area of the graft. Osterix-positive cells observed at 1 month within the PA suggested that the periosteum was a source of osteoprogenitor elements. Alkaline phosphatase data on matrix deposition confirmed this observation.

The present model allowed for a standardized investigation of distinct graft-to-host interfaces both at vertically augmented and inlay-augmented sites, thus possibly limiting the number of animals required for pre-clinical investigations.