## 4 Discussion
In addition, some systematic reviews have reported promising findings regarding the ability of PRF alone or mixed with other grafting materials to preserve bone volume in ARP procedures [11; 22]. Cigarette smoking is a significant factor that inhibits bone healing, as it can generate heat and contains multiple toxic chemical products such as nicotine, a potent vasoconstrtor; carbon monoxide, which can decrease oxygen levels; and hydrogen cyanide, which can cause tissue hypoxia [40], thus reducing the blood supply to the graft area, affecting bone remodeling, and jeopardizing the maintenance of bone volume for future implant placement [30; 41]. Smoking can also decrease the levels of active vitamin D and calcium, thereby impairing normal hard tissue healing [42]. It has also been shown to negatively affect the mRNA expression of bone sialoprotein (BSP) and osteoclast (OCN) and increase the expression of type I collagen (COL-I) [43]. Smoking has also been shown to mutate fibroblasts, altering their differentiation and migration [31], and might have a deleterious effect on wound healing after surgery and decrease regeneration, hence increasing implant failure rates and negatively affecting periodontal regenerative surgeries [30; 44]. Platelet-rich fibrin has shown great promise as a grafting material, as it has a positive effect on vital bone regeneration. It enhances osteoblast proliferation and cell migration as well as increases the expression of multiple genes related to bone formation, including run-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteoclast (OCN) [45]. PRF has also shown, in multiple clinical studies, the ability to improve alveolar bone healing, resulting in better preservation of the alveolar ridge volume when compared to an empty socket [46; 47]. Besides its benefits in ARP procedures, PRF has been shown to improve bone formation in maxillary sinus augmentation and make extensive bone regeneration more predictable [48; 49]. Given PRF's numerous benefits in bone healing, it may help to mitigate the adverse effects of toxins generated from cigarette smoking since it has already been shown to improve the healing of soft tissue [32]. This can be confirmed by the present study's findings, as the A/S-PRF group showed the complete formation of vital bone, while FDBA/CM showed areas of non-vital bone. Some limitations of the present study should be emphasized. First, a stent was not used while taking CBCT images for standardization, as radiopaque objects may have hindered radiographic interpretation. To reduce this limitation, all the images were taken by the same experienced technician using one device; the voltage, current, exposure time and detection field were kept constant for every image taken, and all the measurements were made after superimposing the before and after images using fixed anatomical landmarks [50]. Second, this study did not follow a split-mouth design, and the participant's healing capabilities may influence the outcome; to minimize this limitation, the study was conducted in a randomized fashion. Third, the sample size, although larger than that found in similar studies, is still considered small. Fourth, bone harvesting was postponed after six months from grafting due to the expected impaired healing of smokers, but this may have influenced the histological findings presented in the study, as a longer healing time could result in more vital bone [51]. Fifth, due to the nature of the study, it was not possible for the surgeon to be blinded during the grating; however, blinding was established during the radiographic interpretation and histological analysis to eliminate any bias. Lastly, although RCP was used as a negative control, it cannot be considered a true negative control (i.e., unaided socket healing).