This study aimed to evaluate the use of Bio-Oss, Cerabone, and i-PRF, alone or in combination, in critical size bone defects in rat calvaria. To the authors' best knowledge, no study in the literature has been found so far, in animals or humans, that compares and associates the materials cited in this study.
The application of BO and CB isolated in the critical defects created for this study did not show a statistical difference between them (Table 1). Even more, the BO group presented a lower result during the mean of periods if we consider that there was no difference (p > 0.05) with our control group, CG, differently from CB (p < 0.05). This result contradicts many studies that represent Bio-Oss as the most commonly used bone substitute with good clinical and histological outcomes that favor its use [8, 39, 40]. However, a similar study of Grossi-Oliveira, Faverani, Mendes, Braga Polo, Batista Mendes, de Lima, Ribeiro Junior, Okamoto and Magro-Filho  also reported other graft substitute materials, xenogeneic and alloplastic origin, with better results than the Bio-Oss itself. This could be justified by the slow and incomplete resorption rate of its particles, even exhibiting a superior rate of calcium release as compared to Cerabone as described by the literature [41, 42].
Mahesh, Mascarenhas, Bhasin, Guirado and Juneja  also investigated new bone formation with Bio Oss or Cerabone in sinus augmentation procedures by histologic and histomorphometric analysis. Compared to our results, the use of the xenografts isolated also did not show a statistical difference between the groups but allowed new bone formation in the grafted sinuses, which have been considered both as predictable materials to apply in different clinical procedures. Furthermore, according to the authors, both materials show a similar healing process involving minimal inflammation and resulting in long-term success.
Tawil, Barbeck, Unger, Tawil and Witte  demonstrated that the use of Cerabone in patients submitted to maxillary sinus lift procedures, followed by the installation of implants, proved to be an adequate osteoconductive material with slow and effective resorption. Further, other human studies, where Cerabone and Bio-Oss were compared to assess the bone regeneration process did not observe any statistical difference between the analyzed groups and both xenografts proved to be effective in sinus enlargement of atrophic maxillae . Riachi, Naaman, Tabarani, Aboelsaad, Aboushelib, Berberi and Salameh  also reported, through radiographic analysis of maxillary grafted sinuses, that Bio-Oss presented a significantly greater volumetric loss (33.4 ± 3.1%) than the initial graft size compared to Cerabone (23.4 ± 3.6%), being the largest amount of vertical loss of volume observed after one year of surgery.
The use of i-PRF has been proposed to agglutinate the particulated bone graft material in defects and to enhance its osteoconductive capacity . As explored by many studies in the last decade, the advantages of i-PRF carrying cytokines and growth factors, accelerate cell migration, neovascularization, and the inflammatory response, favoring tissue repair [38, 46]. Furthermore, its capacities can be extrapolated to many periodontal and surgical procedures, proving to be versatile, effective, and of low-cost obtention material.
Similar in vivo studies filling and associating i-PRF to DBBM grafts in calvarial defects are still scarce in the literature. Mu, He, Xin, Li, Yuan, Zou, Shu, Song, Huang and Chen  investigated the angiogenic and osteogenic capacity of DBBM particles soaked in i-PRF for sinus grafting, in rabbits. In this study, i-PRF + DBBM accelerated vascular formation, bone remodeling, and substitution of bone graft materials at the early healing period, even though it failed to increase the bone volume in a long-term period, but demonstrating great potential in the application for sinus augmentations. Using L-PRF in calvarial defects, do Lago, Ferreira, Garcia, Okamoto and Mariano , also exhibited that the association between Bio-Oss and L-PRF showed improvement in bone repair compared to the isolated application of the materials. Also confirming the potential of the platelet concentrates as described by recent studies.
Thus, these studies also suggest that the i-PRF used alone is already capable of promoting bone regeneration [48–50], a situation also observed in the present study, in which the i-PRF was superior to the CG group and statistically similar to the other groups.
In this study, only the AG particles were measured and included in the percentage calculation of new bone formation within the total area of the defect. DBBMs were not counted in the surgical defects. Thus, autogenous bone showed statistical difference from all groups, presenting numerical results considerably higher.
Following the ARRIVE and the Ethics Committee for studies with animals’ guidelines from the Federal University of Alfenas, the required blood volume and collection for the i-PRF preparation in rats is considered a terminal procedure . Therefore, homogenous i-PRF was chosen, since by using donor rats, it was possible to obtain a greater volume of i-PRF . Furthermore, as a possible concern due to this choice, there are reports of the use of donor rats to obtain platelet concentrates without interfering with their results . The risk of antigenicity seems to be insignificant when the donor animals are healthy, of the same age and species as the recipient animals . Thus, it can be suggested that the use of donor animals to obtain blood derivatives in experimental research with small models is feasible . In the present study, no antigenicity reaction was noted in the recipient rats.
Even though the scientific literature suggests an 8-week period for histological analysis of new bone formation in experimental models [55, 56], there was no statistical difference between groups during the 4-week and 8-week periods in this study. Conversely, some studies also suggest that, it is important to evaluate the effects of grafting on bone regeneration after 60 days postoperatively. According to Sohn, Heo, Kwak, Kim, Kim, Moon, Lee and Park , it would be a period long enough to observe the action of the material, evaluating the incorporation, remodeling, and resorption of the graft. Therefore, to confirm the capacity of new bone regeneration with the materials used in our study, a period longer than 8 weeks could confirm the study of Sohn, Heo, Kwak, Kim, Kim, Moon, Lee and Park .
Finally, the scarcity of studies in the literature with the same study model for the comparison of different substitute materials, as performed by this study, is highlighted. This justifies its importance and highlights the need for further investigations into different bone substitutes and platelet concentrates with more homogeneous methodologies. Furthermore, the structural and financial limitations in the period and location of the same study limited the performance of more refined analyzes that could provide even more understanding about the proposed investigation.